Methods for reducing intraocular pressure by administering a soluble hyaluronidase glycoprotein (sHASEGP)

ABSTRACT

Provided are methods for reducing intraocular pressure in the eye of a subject by administering neutral active soluble active hyaluronidase glycoproteins (sHASEGPs) and modified forms thereof to the eye. The sHASEGPs are soluble forms of human PH20 hyaluronidase. Also provided are the sHASEGPs, methods of manufacture, and uses thereof. Minimally active polypeptide domains of the soluble, neutral active sHASEGP domains are described. The sHASEGPs include asparagine-linked sugar moieties required for a functional neutral active hyaluronidase domain.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of previously U.S. application Ser.No. 13/694,005, filed Oct. 18, 2012, now issued U.S. Pat. No. 8,772,246,entitled “SOLUBLE HYALURONIDASE GLYCOPROTEIN (sHASEGP), PROCESS FORPREPARING THE SAME, USES AND PHARMACEUTICAL COMPOSITIONS COMPRISINGTHEREOF,” which is a continuation of U.S. application Ser. No.12/378,984, now U.S. Pat. No. 8,431,380, entitled “SOLUBLE HYALURONIDASEGLYCOPROTEIN (sHASEGP), PROCESS FOR PREPARING THE SAME, USES ANDPHARMACEUTICAL COMPOSITIONS COMPRISING THEREOF,” filed Feb. 20, 2009,which is a continuation of U.S. application Ser. No. 10/795,095, nowU.S. Pat. No. 7,767,429, entitled “SOLUBLE HYALURONIDASE GLYCOPROTEIN(sHASEGP), PROCESS FOR PREPARING THE SAME, USES AND PHARMACEUTICALCOMPOSITIONS COMPRISING THEREOF,” filed Mar. 5, 2004, which claims thebenefit of priority under 35 U.S.C. §119(e) to U.S. provisionalApplication Ser. No. 60/452,360, filed Mar. 5, 2003, each to LouisBookbinder, Anirban Kundu and Gregory I. Frost.

Benefit of priority under 35 U.S.C. §120 is claimed to each of theabove-noted applications and patents. The subject matter of each of theabove-noted U.S. applications and patents is incorporated in itsentirety by reference thereto.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is generally related to Neutral-Active, SolubleHyaluronidase Glycoproteins (sHASEGP), and portions thereof,particularly Hyaluronidase domains. More specifically, the invention isrelated to chemical modifications, pharmaceutical compositions,expression plasmids, methods for manufacture and therapeutic methodsusing the Hyaluronidase Glycoproteins and domains thereof and theencoding nucleic acid molecules for the therapeutic modification ofglycosaminoglycans in the treatment of disease and for use to increasediffusion of other injected molecules less than 200 nanometers indiameter in an animal.

Background Information

Glycosaminoglycans (GAGs) are complex linear polysaccharides of theextracellular matrix (ECM). GAG's are characterized by repeatingdisaccharide structures of an N-substituted hexosamine and a uronicacid, [hyaluronan (HA), chondroitin sulfate (CS), chondroitin (C),dermatan sulfate (DS), heparan sulfate (HS), heparin (H)], or agalactose [keratan sulfate (KS)]. Except for HA, all exist covalentlybound to core proteins. The GAGs with their core proteins arestructurally referred to as proteoglycans (PGs).

Hyaluronan (HA) is found in mammals predominantly in connective tissues,skin, cartilage, and in synovial fluid. Hyaluronan is also the mainconstituent of the vitreous of the eye. In connective tissue, the waterof hydration associated with hyaluronan creates spaces between tissues,thus creating an environment conducive to cell movement andproliferation. Hyaluronan plays a key role in biological phenomenaassociated with cell motility including rapid development, regeneration,repair, embryogenesis, embryological development, wound healing,angiogenesis, and tumorigenesis (Toole 1991 Cell Biol. Extracell.Matrix, Hay (ed), Plenum Press, New York, 1384-1386; Bertrand et al.1992 Int. J. Cancer 52:1-6; Knudson et al., 1993 FASEB J. 7:1233-1241).In addition, hyaluronan levels correlate with tumor aggressiveness(Ozello et al. 1960 Cancer Res. 20:600-604; Takeuchi et al. 1976, CancerRes. 36:2133-2139; Kimata et al. 1983 Cancer Res. 43:1347-1354).

HA is found in the extracellular matrix of many cells, especially insoft connective tissues. HA has been assigned various physiologicalfunctions, such as in water and plasma protein homeostasis (Laurent T Cet al. (1992) FASEB J 6: 2397-2404). HA production increases inproliferating cells and may play a role in mitosis. It has also beenimplicated in locomotion and cell migration. HA seems to play importantroles in cell regulation, development, and differentiation (Laurent etal., supra).

HA has been used in clinical medicine. Its tissue protective andrheological properties have proved useful in ophthalmic surgery toprotect the corneal endothelium during cataract surgery. Serum HA isdiagnostic of liver disease and various inflammatory conditions, such asrheumatoid arthritis. Interstitial edema caused by accumulation of HAmay cause disfunction in various organs (Laurent et al., supra).

Hyaluronan protein interactions also are involved in the structure ofthe extracellular matrix or “ground substance”.

Hyaluronidases are a group of neutral- and acid-active enzymes foundthroughout the animal kingdom. Hyaluronidases vary with respect tosubstrate specificity, and mechanism of action.

There are three general classes of hyaluronidases:

1. Mammalian-type hyaluronidases, (EC 3.2.1.35) which areendo-beta-N-acetylhexosaminidases with tetrasaccharides andhexasaccharides as the major end products. They have both hydrolytic andtransglycosidase activities, and can degrade hyaluronan and chondroitinsulfates (CS), specifically C4-S and C6-S.

2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan and, and tovarious extents, CS and DS. They are endo-beta-N-acetylhexosaminidasesthat operate by a beta elimination reaction that yields primarilydisaccharide end products.

3. Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, andcrustaceans are endo-beta-glucuronidases that generate tetrasaccharideand hexasaccharide end products through hydrolysis of the beta 1-3linkage.

Mammalian hyaluronidases can be further divided into two groups: neutralactive and acid active enzymes. There are six hyaluronidase-like genesin the human genome, HYAL1, HYAL2, HYAL3 HYAL4 HYALP1 and PH20/SPAM1.HYALP1 is a pseudogene, and HYAL3 has not been shown to possess enzymeactivity toward any known substrates. HYAL4 is a chondroitinase andlacks activity towards hyaluronan. HYAL1 is the prototypical acid-activeenzyme and PH20 is the prototypical neutral-active enzyme. Acid activehyaluronidases, such as HYAL1 and HYAL2 lack catalytic activity atneutral PH. For example, HYAL1 has no catalytic activity in vitro overpH 4.5 (Frost et al., Anal Biochemistry, 1997). HYAL2 is an acid activeenzyme with a very low specific activity in vitro.

The hyaluronidase-like enzymes can also be characterized by those whichare locked to the plasma membrane via a glycosylphosphatidyl inositolanchor such as human HYAL2 and human PH20 (Danilkovitch-Miagkova et al.,Proc Natl Acad Sci USA. 2003 Apr. 15; 100(8):4580-5, Phelps et al.,Science 1988) and those which are soluble such as human HYAL1 (Frost etal., Biochem Biophys Res Commun. 1997 Jul. 9; 236(1):10-5). However,there are variations from species to species: bovine, PH20 for exampleis very loosely attached to the plasma membrane and is not anchored viaa phospholipase sensitive anchor (Lalancette et al., Biol Reprod. 2001August; 65(2):628-36.). This unique feature of bovine hyaluronidase haspermitted the use of the soluble bovine testes hyaluronidase enzyme asan extract for clinical use (Wydase®, Hyalase®). Other PH20 species arelipid anchored enzymes that are not insoluble without the use ofdetergents or lipases. For example, human PH20 is anchored to the plasmamembrane via a GPI anchor. Attempts to make human PH20 DNA constructsthat would not introduce a lipid anchor into the polypeptide resulted ineither a catalytically inactive enzyme, or an insoluble enzyme (Arminget al., Eur J Biochem. 1997 Aug. 1; 247(3):810-4). Naturally occurringmacaque sperm hyaluronidase is found in both a soluble and membranebound form. While the 64 kDa membrane bound form possesses enzymeactivity at pH 7.0, the 54 kDa form is only active at pH 4.0 (Cherr etal., Dev Biol. 1996 Apr. 10; 175(1):142-53.). Thus, soluble forms ofPH20 are often lacking enzyme activity under neutral conditions.

Chondroitinases are enzymes found throughout the animal kingdom. Theseenzymes degrade glycosaminoglycans through an endoglycosidase reaction.Specific examples of known Chondroitinases include Chondroitinase ABC(derived from Proteus vulgaris; Japanese Patent Application Laid-open No6-153947, T. Yamagata, H. Saito, O. Habuchi, and S. Suzuki, J. Biol.Chem., 243, 1523 (1968), S. Suzuki, H. Saito, T. Yamagata, K. Anno, N.Seno, Y. Kawai, and T. Furuhashi, J. Biol. Chem., 243, 1543 (1968)),Chondroitinase AC (derived from Flavobacterium heparinum; T. Yamagata,H. Saito, O. Habuchi, and S. Suzuki, J. Biol. Chem., 243, 1523 (1968)),Chondroitinase AC II (derived from Arthrobacter aurescens; K. Hiyama,and S. Okada, J. Biol. Chem., 250, 1824 (1975), K. Hiyama and S. Okada,J. Biochem. (Tokyo), 80, 1201 (1976)), Hyaluronidase ACIII (derived fromFlavobacterium sp. Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, KeiichiYoshida, Kiyoshi Morikawa, and Kiyochika Tokuyasu, Seikagaku, 61, 1023(1989)), Chondroitinase B (derived from Flavobacterium heparinum; Y. M.Michelacci and C. P. Dietrich, Biochem. Biophys. Res. Commun., 56, 973(1974), Y. M. Michelacci and C. P. Dietrich, Biochem. J., 151, 121(1975), Kenichi Maeyama, Akira Tawada, Akiko Ueno, and Keiichi Yoshida,Seikagaku, 57, 1189 (1985)), Chondroitinase C (derived fromFlavobacterium sp. Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, KelichiYoshida, Kiyoshi Morikawa, and Kiyochika Tokuyasu, Seikagaku, 61, 1023(1939)), and the like.

Glycoproteins are composed of a polypeptide chain covalently bound toone or more carbohydrate moieties. There are two broad categories ofglycoproteins that possess carbohydrates coupled though eitherN-glycosidic or O-glycosidic linkages to their constituent protein. TheN- and O-linked glycans are attached to polypeptides throughasparagine-N-acetyl-D-glucosamine and serine(threonine)-N-acetyl-D-galactosamine linkages, respectively. ComplexN-linked oligosaccharides do not contain terminal mannose residues. Theycontain only terminal N-acetylglucosamine, galactose, and/or sialic acidresidues. Hybrid oligosaccharides contain terminal mannose residues aswell as terminal N-acetylglucosamine, galactose, and/or sialic acidresidues.

With N-linked glycoproteins, an oligosaccharide precursor is attached tothe amino group of asparagine during peptide synthesis in theendoplasmic reticulum. The oligosaccharide moiety is then sequentiallyprocessed by a series of specific enzymes that delete and add sugarmoieties. The processing occurs in the endoplasmic reticulum andcontinues with passage through the cis-, medial- and trans-Golgiapparatus.

SUMMARY OF THE INVENTION

Provided herein are members of the soluble, neutral active HyaluronidaseGlycoprotein family, particularly the human soluble PH-20 HyaluronidaseGlycoproteins (also referred to herein as sHASEGPs). The sHASEGPprovided herein is a sHASEGP family member, designated herein as asHASEGP. The soluble Hyaluronidase domain, and uses thereof, are alsoprovided.

The invention is based upon the discovery that a soluble, neutral-activehyaluronidase activity can be produced with high yield in a mammalianexpression system by introducing nucleic acids that lack a narrow regionencoding amino acids in the carboxy terminus of the human PH20 cDNA.Additional modifications of the sHASEGP to enhance secretion by use ofnon-native leader peptides are also provided. Further provided aremethods to modify the sHASEGP to prolong its half life by way of maskingthe protein with polyethylene glycol and posttranslational modificationsto native glycosylation. Previous attempts to generate secreted aneutral active human sHASEGP were unsuccessful. It was concluded thattruncations of the human sHASEGP polypeptide resulted in both a loss ofneutral enzymatic activity, and an inability of cells to secrete therecombinant protein in mammalian expression systems (Arming et al., EurJ Biochem 1997 Aug. 1; 247 (3):810-4). It is critical to generateneutral-acting secreted sHASEGP for commercial production andtherapeutic utility as a hyaluronidase. The invention, disclosed herein,overcomes such challenges.

The invention further comprises a catalytically active human sHASEGPglycoprotein wherein the sHASEGP possesses at least one N-linked sugarmoiety. The studies shown herein demonstrate that human PH20 requiresN-linked glycans for catalytic activity, whereas bovine and bee venomhyaluronidases remain active without such N-linked glycans. A humanhyaluronidase domain devoid of N-linked moieties is catalyticallyinactive. Thus classic recombinant DNA technology does not permit theproduction of a catalytically active human sHASEGP, unlike bee venomHASEGP, which can be produced in e. coli.

The invention includes methods and cells for generation of an N-linkedsHASEGP glycoprotein polypeptide, by using of a cell capable ofintroducing said N-linked sugar moieties or by introduction of saidN-linked moieties on a sHASEGP polypeptide. Methods of identifyingproperly glycosylated sHASEGP's are further disclosed.

Catalytically active Super-Sialated sHASEGP glycoproteins are alsoprovided. Super-sialated sHASEGPs possess greater serum half-livescompared to naturally occurring non-sialated bovine and ovine testessHASEGPs, and are thus preferable for both enzyme stability and use asintravenous drugs. The invention provides methods for the preparation ofSuper-Sialated sHASEGPs, compositions and uses thereof.

Proteins encoded by naturally GPI anchor deficient sHASEGP's splicevariants are also provided.

Further provided are compositions of the sHASEGP comprising, a solublesHASEGP glycoprotein with a metal ion, wherein the metal ion is Calcium,Magnesium or Sodium. sHASEGPs are optimally active in the presence ofsaid metals. Formulations consisting of sHASEGP in the presence of saidmetal ions are also provided.

Modifications of sHASEGP to further prolong the half life are provided.Chemical modifications of a sHASEGP with polymers such as polyethyleneglycol and dextran are provided. Such modifications shield sHASEGP'sfrom removal from circulation and the immune system as well asglycosylation receptors for mannose and asialoglycoprotein. Furtherprovide are methods to link to specific functional groups such asglycosylation sites, positively charged amino acids and cysteines.

Assays for identifying effectors, such as compounds, including smallmolecules, and conditions, such pH, temperature and ionic strength, thatmodulate the activation, expression or activity of sHASEGP are alsoprovided herein. In exemplary assays, the effects of test compounds onthe ability of a Hyaluronidase domain of sHASEGP to cleave a knownsubstrate, typically a glycosaminoglycan or proteoglycan, are assessed.Agents, generally compounds, particularly small molecules, that modulatethe activity of the Hyaluronidase domain are candidate compounds formodulating the activity of the sHASEGP. The Hyaluronidase domains canalso be used to produce Hyaluronidase-specific antibodies with functionperturbing activity. The Hyaluronidase domains provided herein include,but are not limited to, the N-terminal glycsoyl-hydrolase domain withC-terminal truncated portions thereof that exhibit catalytic activity invitro.

Nucleic acid molecules encoding the proteins and Hyaluronidase domainsare also provided. Nucleic acid molecules that encode a solubleHyaluronidase domain or catalytically active portions thereof and alsothose that encode the full-length sHASEGP are provided. Nucleic acidencoding the Hyaluronidase domain and downstream nucleic acid is setforth in SEQ ID No. 6; and the Hyaluronidase domain of sHASEGP is setforth in SEQ ID No. 1 (amino acids 35-464). The protein sequence andencoding nucleic acid sequence of the full-length sHASEGP are set forthin SEQ ID Nos. 1 and 6.

Also provided are nucleic acid molecules that hybridize to suchsHASEGP-encoding nucleic acid along their full-length or along at leastabout 70%, 80% or 90% of the full-length and encode the Hyaluronidasedomain or portion thereof are provided. Hybridization is generallyeffected under conditions of at least low, generally at least moderate,and often high stringency.

The isolated nucleic acid fragment is DNA, including genomic or cDNA, oris RNA, or can include other components, such as protein nucleic acid orother nucleotide analogs. The isolated nucleic acid may includeadditional components, such as heterologous or native promoters, andother transcriptional and translational regulatory sequences, thesegenes may be linked to other genes, such as reporter genes or otherindicator genes or genes that encode indicators.

Also provided is an isolated nucleic acid molecule that includes thesequence of molecules that is complementary to the nucleotide sequenceencoding sHASEGP or the portion thereof.

Also provided are fragments thereof or oligonucleotides that can be usedas probes or primers and that contain at least about 10, 14, 16nucleotides, generally less than 1000 or less than or equal-to 100, setforth in SEQ ID NO. 6 (or the complement thereof); or contain at leastabout 30 nucleotides (or the complement thereof) or containoligonucleotides that hybridize along their full-length (or at leastabout 70, 80 or 90% thereof) to any such fragments or oligonucleotides.The length of the fragments are a function of the purpose for which theyare used and/or the complexity of the genome of interest. Generallyprobes and primers contain less than about 50, 150 or 500 nucleotides.

Also provided are plasmids containing any of the nucleic acid moleculesprovided herein. Cells containing the plasmids are also provided. Suchcells include, but are not limited to, bacterial cells, yeast cells,fungal cells, plant cells, insect cells and animal cells.

Also provided are enhanced mammalian expression systems using signalleaders capable of efficient secretion of sHASEGP. An example of suchefficient secretory leader peptide amino acid sequence and fusionprotein with sHASEGP is found in SEQ ID Nos. 43 and 46.

Also provided is a method of producing sHASEGP by growing theabove-described cells under conditions whereby the sHASEGP is expressedby the cells, and recovering the expressed sHASEGP polypeptide orglycoprotein. Methods for isolating nucleic acid encoding other sHASEGPsare also provided.

Also provided are cells, generally eukaryotic cells, such as mammaliancells and yeast cells, in which the sHASEGP polypeptide is expressed onthe surface of the cells. Such cells are used in drug screening assaysto identify compounds that modulate the activity of the sHASEGPpolypeptide. These assays, including in vitro binding assays, andtranscription based assays in which signal transduction mediateddirectly or indirectly, such as via activation of pro-growth factors, bythe sHASEGP is assessed.

Also provided are peptides encoded by such nucleic acid molecules.Included among those polypeptides is the sHASEGP Hyaluronidase domain ora polypeptide with amino acid changes such that the specificity and/orHyaluronidase activity remains substantially unchanged. In particular, asubstantially purified mammalian sHASEGP glycoprotein is provided thatincludes a secreted neutral catalytically active

The invention also includes a Hyaluronidase catalytic domain and mayadditionally include other domains. The sHASEGP may form homodimers andcan also form heterodimers with some other protein, such as amembrane-bound protein. Also provided is a substantially purifiedglycoprotein including a sequence of amino acids that has at least 60%,70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sHASEGP where thepercentage identity is determined using standard algorithms and gappenalties that maximize the percentage identity.

Splice variants of the sHASEGP, particularly those with a catalyticallyactive Hyaluronidase domain, are contemplated herein.

In other embodiments, substantially purified polypeptides that include aHyaluronidase domain of a sHASEGP polypeptide or a catalytically activeportion thereof, but that do not include the entire sequence of aminoacids set forth in SEQ ID No. 1 are provided. Among these arepolypeptides that include a sequence of amino acids that has at least70%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID No. 1 or 3.

In a specific embodiment, a nucleic acid that encodes a eukaryoticHyaluronidase glycoprotein, designated sHASEGP is provided. Inparticular, the nucleic acid includes the sequence of nucleotides setforth in SEQ ID No. 6, particularly set forth as nucleotides 106-1446 ofSEQ ID NO. 6, or a portion there of that encodes a catalytically activepolypeptide.

Also provided are nucleic acid molecules that hybridize under conditionsof at least low stringency, generally moderate stringency, moretypically high stringency to the SEQ ID NO. 6 or degenerates thereof.

In one embodiment, the isolated nucleic acid fragment hybridizes to anucleic acid molecule containing the nucleotide sequence set forth inSEQ ID No: 6 (or degenerates thereof) under high stringency conditions.A full-length sHASEGP is set forth in SEQ ID No. 1 and is encoded by SEQID NO. 6 or degenerates thereof.

Also provided are muteins of the Hyaluronidase domain of sHASEGP,particularly muteins in which the Cys residue in the Hyaluronidasedomain that is free i.e., does not form disulfide linkages with anyother Cys residue in the Hyaluronidase domain) is substituted withanother amino acid substitution, typically, although not necessarily,with a conservative amino acid substitution or a substitution that doesnot eliminate the activity, and muteins in which a specificglycosylation site (s) is eliminated.

sHASEGP polypeptides, including, but not limited to splice variantsthereof, and nucleic acids encoding sHASEGPs, and domains, derivativesand analogs thereof are provided herein. Single chain secretedHyaluronidase glycoproteins that have an N-terminus functionallyequivalent to that generated by activation of a signal peptidase to formsHASEGP are also provided. There are seven potential N-linkedglycosylation sites at N82, N166, N235, N254, N368, N393, N490 ofsHASEGP as exemplified in SEQ ID NO: 1. Disulfide bonds form between theCys residues C60-C351 and Cys residues C224 to C238 to form the coreHyaluronidase domain. However, additional cysteines are required in thecarboxy terminus for neutral enzyme catalytic activity such that sHASEGPfrom amino acids 36 to Cys 464 in SEQ ID No.1 comprise the minimallyactive human sHASEGP hyaluronidase domain. Thus, N-linked glycosylationsite N-490 is not required for proper sHASEGP activity.

N-linked glycosylation of the sHASEGP's are critical for their catalyticactivity and stability. While altering the type of glycan modifying aglycoprotein can have dramatic affects on a protein's antigenicity,structural folding, solubility, and stability, most enzymes are notthought to require glycosylation for optimal enzyme activity. sHASEGPsare thus unique in this regard, that removal of N-linked glycosylationcan result in near complete inactivation of the Hyaluronidase activity.The presence of N-linked glycans is critical for generating an activesHASEGP. Protein expression systems suitable for the introduction ofcritical N-linked glycosylation residues on sHASEGP are included.Additionally, the introduction of deglycosylated sHASEGP polypeptides inthe presence of extracts capable of introducing N-linked glycans isincluded. In one aspect of the invention, complex glycosylation cappedwith sialation is described whereas others capped with free mannoseresidues are contemplated as well. Preferably, sialic acid residues arefound in the terminal residues of N-linked glycosylation on sHASEGP.

N-linked oligosaccharides fall into several major types (oligomannose,complex, hybrid, sulfated), all of which have (Man)3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn residuesthat fall within -Asn-Xaa-Thr/Ser-sequences (where Xaa is not Pro).Glycosylation at an -Asn-Xaa-Cys-site has been reported for coagulationprotein C. N-linked sites are often indirectly assigned by theappearance of a “blank” cycle during sequencing. Positive identificationcan be made after release of the oligosaccharide by PNGase F, whichconverts the glycosylated Asn to Asp. After PNGase F release, N-linkedoligosaccharides can be purified using Bio-Gel P-6 chromatography, withthe oligosaccharide pool subjected to preparative high pH anion exchangechromatography (HPAEC) (Townsend et al., (1989) Anal. Biochem. 182,1-8). Certain oligosaccharide isomers can be resolved using HPAEC.Fucose residues will shift elution positions earlier in the HPAECchromatogram, while additional sialic acid residues will increase theretention time. Concurrent treatment of glycoproteins whoseoligosaccharide structures are known (e.g., bovine fetuin, a-l acidglycoprotein, ovalbumin, RNAse B, transferrin) can facilitate assignmentof the oligosaccharide peaks. The collected oligosaccharides can becharacterized by a combination of compositional and methylation linkageanalyses (Waegheet al., (1983) Carbohydr Res. 123, 281-304.), withanomeric configurations assigned by NMR spectroscopy (Van Halbeek (1993)in Methods Enzymol 230).

Formulations of sHASEGP's are also provided. sHASEGPs may be formulatedin lyophilized forms and stabilized solutions. Formulations containingspecific metal ions, such as calcium, magnesium, or sodium, are usefulfor optimal activity at neutral PH. In addition to stabilized solutionformulations, slow release formulations are contemplated herein forextended removal of glycosaminoglycans. Also provided herein are kitsproviding for pre-packaged syringes of sHASEGP's for the administrationof small volumes of sHASEGP for intraocular surgical procedures andother small volume procedures. Balanced salt formulations for ex vivouse in artificial reproductive technology procedures are also provided.

Methods for the use of sHASEGP's in the removal of glycosaminoglycansare also provided. sHASEGPs open channels in the interstitial spacethrough degradation of glycosaminoglycans that permit the diffusion ofmolecules less than 500 nm in size. These channels remain for a periodof 24-48 hours depending on dose and formulation. Such channels can beused to facilitate the diffusion of exogenously added molecules such asfluids, small molecules, proteins, nucleic acids and gene therapyvectors and other molecules less than 500 nm in size.

sHASEGPs can also be used to remove excess glycosaminoglycans such asthose that occur following ischemia reperfusion, inflammation,arteriosclerosis, edema, cancer, spinal cord injury and other forms ofscarring. In some instances, sHASEGP's can be delivered systemically byintravenous infusion. This can be helpful when local access is notreadily available such as the heart or brain or in the case ofdisseminated neoplasm wherein the disease is through the body.Super-Sialated sHASEGP's are preferable to increase serum half-life anddistribution over native hyaluronidase enzymes that lack terminal sialicacids.

In some circumstances, such as spinal cord injury, glaucoma, andcosmetic treatments, sustained delivery is preferred.

In other indications, a single short acting dose is preferable.Temporary removal of glycosaminoglycans can be used to enhance thedelivery of solutions and drugs into interstitial spaces. This can beuseful for the diffusion of anesthesia and for the administration oftherapeutic fluids, molecules and proteins. Subcutaneous andIntramuscular administration of molecules in the presence of sHASEGP'salso facilitate their systemic distribution more rapidly. Such methodsare very useful when intravenous access is not available or where morerapid systemic delivery of molecules is needed. Delivery of other largemolecules such as Factor VIII, that are poorly bioavailable uponsubcutaneous administration, may be injected with sHASEGP's to increasetheir availability.

Uses of sHASEGP's for enzymatic removal of the cumulus matrixsurrounding oocytes are also provided. The removal of the cumulus matrixusing a purified sHASEGP without the toxic contaminants of extractderived hyaluronidase permits more gentle recover of the oocyte withgreater viabilities. Moreover, sHASEGP's can be manufactured without theuse of cattle extracts or other organisms that carry viruses and otherpathogens such as transmissible spongiform ecephalopathies.

Injections of small volumes of sHASEGP for intraocular use may also beused for small spaces. SHASEGPs may be injected into the anteriorchamber of the eye to remove excess viscoelastic substrates that areadministered during surgery. Intraocular injection of sHASEGP's can alsobe used to reduce intraocular pressure in glaucoma, to dissolve vitreousaggregates, or “floaters”, to clear vitreous hemorrhage, for thetreatment of macular degeneration, to promote vitreo retinal detachmentin diabetic retinopathy and to be mixed with other enzymes to promotereshaping of the cornea along with corrective lenses. It will berecognized that in some instances, the use of a long lasting sHASEGPsuch as a pegylated-sHASEGP will be desirable.

Co-formulations of sHASEGP with other substances may also be envisionedfor injectable pens for small volume or rapid subcutaneousadministration. Examples such as Epipen®, insulin, and other fluids canbe formulated. The methods of the invention include administration ofthe sHASEGP polypeptide or pharmaceutical compositions containingsHASEGP prior to, simultaneously with or following administration ofother therapeutic molecules. The sHASEGP may be administered at a sitedifferent from the site of administration of the therapeutic molecule orthe sHASEGP may be administered at a site the same as the site ofadministration of the therapeutic molecule.

Hence, provided herein is a family of eukaryotic secreted neutral activehyaluronidase glycoproteins designated sHASEGP's, and functionaldomains, especially Hyaluronidase (or catalytic) domains thereof,muteins and other derivatives and analogs thereof. Also provided hereinare nucleic acids encoding the sHASEGPs. Additionally provided areformulations and therapeutic uses of said sHASEGP's to treat disease andfor use as tissue modifying enzymes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a vector map of sHASEGP Vector HZ24.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention (s) belong. All patents, patent applications,published applications and publications, Genbank sequences, websites andother published materials referred to throughout the entire disclosureherein, unless noted otherwise, are incorporated by reference in theirentirety. In the event that there is a plurality of definitions forterms herein, those in this section prevail.

Where reference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:942-944).

As used herein, eukaryotic Hyaluronidase refers to a diverse family ofglycosaminoglycan endoglucosaminidases, wherein a glutamate residue inthe Hyaluronidase hydrolyzes the beta 1,4 linkages of hyaluronan andchondroitin sulfates through an acid-base catalytic mechanism.

Of particular interest are sHASEGP's of mammalian, including human,origin. Those of skill in this art recognize that, in general, singleamino acid substitutions in non-essential regions of a polypeptide donot substantially alter biological activity (see, e.g., Watson et al.,(1987) Molecular Biology of the Gene, 4th Edition, The Benjamin/CummingsPub. co., p. 224).

As used herein, membrane anchored sHASEGP, refers to a family ofmembrane anchored Hyaluronidases that share common structural featuresas described herein.

As used herein, soluble hyaluronidase refers to a polypeptidecharacterized by its solubility under physiologic conditions. SolubleHASEGP can be distinguished for example by its partitioning into theaqueous phase of a Triton X-114 solution warmed to 37 C (Bordier et al JBiol Chem. 1981 Feb. 25; 256(4):1604-7). Lipid anchored HASEGP on theother hand will partition into the detergent rich phase, but willpartition into the detergent poor or aqueous phase following treatmentwith Phospholipase-C.

Thus, reference, for example, to “sHASEGP” encompasses all glycoproteinsencoded by the sHASEGP gene family, including but not limited to: HumansHASEGP, mouse sHASEGP, or an equivalent molecule obtained from anyother source or that has been prepared synthetically or that exhibitsthe same activity. Sequences of encoding nucleic acid molecules and theencoded amino acid sequences of exemplary sHASEGP's and/or domainsthereof are set forth, for example in SEQ ID NO: 4. The term alsoencompasses sHASEGP with amino acid substitutions that do notsubstantially alter activity of each member and also encompasses splicevariants thereof. Suitable substitutions, including, although notnecessarily, conservative substitutions of amino acids, are known tothose of skill in this art and can be made without eliminating thebiological activity, such as the catalytic activity, of the resultingmolecule.

As used herein, a sHASEGP, whenever referenced herein, includes at leastone or all of or any combination of: a polypeptide encoded by thesequence of nucleotides set forth in SEQ ID NO. 6 or by a sequence ofnucleotides that includes nucleotides that encode amino acids 1-509 ofSEQ ID No. 1; a polypeptide encoded by a sequence of nucleotides thathybridizes under conditions of low, moderate or high stringency to thesequence of nucleotides set forth in SEQ ID NO. 6; a polypeptide thatincludes the sequence of amino acids set forth as amino acids 1-509 ofSEQ ID No. 1; a polypeptide that includes a sequence of amino acidshaving at least about 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity with the sequence of amino acids set forth in SEQ IDNo. 1 or as amino acids 1-448 of SEQ ID No. 4.

In particular, the sHASEGP polypeptide, with the Hyaluronidase domainsas indicated in SEQ ID No. 4 is provided. The polypeptide is a single ortwo chain polypeptide. Smaller portions thereof that retainHyaluronidase activity are also provided. The Hyaluronidase domains fromsHASEGPs vary in size and constitution, including insertions anddeletions in surface loops. Thus, for purposes herein, the catalyticdomain is a portion of a sHASEGP, as defined herein, and is homologousto a domain of other hyaluronidase like sequences, such as HYAL1, HYAL2,HYAL3, which have been previously identified; it was not recognized,however, that an isolated single chain form of the human Hyaluronidasedomain could function in in vitro assays. The Aspartate and Glutamateresidues necessary for activity are present in conserved motifs.

As used herein, a “neutral hyaluronidase domain of a soluble sHASEGP”refers to an beta 1,4 endoglucosaminidase domain of a sHASEGP thatexhibits Hyaluronidase activity at neutral PH, is soluble underconditions as described and shares homology and structural features withthe hyaluronidase glycosyl-hydrolase family domains but containsadditional sequences in the carboxy terminus that are required forneutral activity. Hence it is at least the minimal portion of the domainthat exhibits Hyaluronidase activity as assessed by standard in vitroassays and remains soluble. Contemplated herein are such Hyaluronidasedomains and catalytically active portions thereof. Also provided aretruncated forms of the Hyaluronidase domain that include the smallestfragment thereof that acts catalytically as a single chain form.

A Hyaluronidase domain of an sHASEGP, whenever referenced herein,includes at least one or all of or any combination of or a catalyticallyactive portion of: an N-linked glycoprotein polypeptide that includesthe sequence of amino acids set forth in SEQ ID No. 1; a polypeptideencoded by a sequence of nucleotides that hybridizes under conditions oflow, moderate or high stringency to the sequence of nucleotides setforth in SEQ ID NO. 6; a polypeptide that includes the sequence of aminoacids set forth in SEQ ID No. 1; a polypeptide that includes a sequenceof amino acids having at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity with the sequence of amino acids set forth in SEQID No. 1; and/or a Hyaluronidase domain of a polypeptide encoded by asplice variant of the sHASEGP.

Thus, for purposes herein, the Hyaluronidase domain is a portion of asHASEGP, as defined herein, and is homologous to a domain of othersHASEGP's. As with the larger class of enzymes of the hyaluronidasefamily, the sHASEGP catalytic domains share a high degree of amino acidsequence identity. The Asp and Glu residues necessary for activity arepresent in conserved motifs.

By active form is meant a form active in vivo and/or in vitro. Asdescribed herein, the Hyaluronidase domain also can exist as a solublesecreted glycoprotein. It is shown herein that, at least in vitro, thesingle chain forms of the sHASEGP's and the catalytic domains orenzymatically active portions thereof (typically C-terminal truncations)exhibit Hyaluronidase activity. Hence provided herein are isolated formsof the Hyaluronidase domains of sHASEGP's and their use in in vitro drugscreening assays for identification of agents that modulate the activitythereof.

As used herein, the catalytically active domain of a sHASEGP refers tothe neutral active endoglucosaminidase domain as defined by activity invitro towards a glycosaminoglycan substrate.

sHASEGPs of interest include those that are active against chondroitinsulfates and chondroitin sulfate proteoglycans (CSPG's) in vivo and invitro; and those that are active against hyaluronan. As used herein, ahuman sHASEGP is one encoded by nucleic acid, such as DNA, present inthe genome of a human, including all allelic variants and conservativevariations as long as they are not variants found in other mammals.

As used herein, nucleic acid encoding a Hyaluronidase domain orcatalytically active portion of a sHASEGP” shall be construed asreferring to a nucleic acid encoding only the recited single chainHyaluronidase domain or active portion thereof, and not the othercontiguous portions of the sHASEGP as a continuous sequence.

As used herein, “disease” or “disorder” refers to a pathologicalcondition in an organism resulting from, e.g., infection or geneticdefect, and characterized by identifiable symptoms.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic nucleic acid,such as DNA, that results in more than one type of mRNA. Splice variantsof sHASEGPs are provided herein.

As used herein, the Hyaluronidase domain of a sHASEGP protein refers tothe Hyaluronidase domain of a sHASEGP that exhibits neutralendoglucosaminidase activity. Hence it is at least the minimal portionof the protein that exhibits endoglucosaminidase activity as assessed bystandard assays in vitro. Exemplary human Hyaluronidase domains includeat least a sufficient portion of sequences of amino acids set forth inSEQ ID No. 4 to exhibit endoglucosaminidase activity.

Also contemplated are nucleic acid molecules that encode a polypeptidethat has endoglucosaminidase activity in an in vitro Hyaluronidase assayand that have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity with the full-length of a Hyaluronidase domain of an sHASEGPpolypeptide, or that hybridize along their full-length or along at leastabout 70%, 80% or 90% of the full-length to a nucleic acids that encodea Hyaluronidase domain, particularly under conditions of moderate,generally high, stringency.

For the Hyaluronidase domains, residues at the in the N-terminal regioncan be critical yet not sufficient for activity. It is shown herein thatthe Hyaluronidase domain of the sHASEGP is catalytically active. Hencethe Hyaluronidase domain generally requires the N-terminal amino acidsthereof for activity; the C-terminus portion can be truncated until thelast Cysteine residue yet requires additional amino acids to beoptimally active. The amount that can be removed can be determinedempirically by testing the polypeptide for Hyaluronidase activity in anin vitro assay that assesses catalytic cleavage.

Hence smaller portions of the Hyaluronidase domains, particularly thesingle chain domains, thereof that retain Hyaluronidase activity arecontemplated. Such smaller versions generally are C-terminal truncatedversions of the Hyaluronidase domains. The Hyaluronidase domains vary insize and constitution, including insertions and deletions in surfaceloops. Such domains exhibit conserved structure, including at least onestructural feature, such as the proton donor, and/or other features ofHyaluronidase domains of endoglucosaminidases. Thus, for purposesherein, the Hyaluronidase domain is a single chain portion of a sHASEGP,as defined herein, but is homologous in its structural features andretention of sequence of similarity or homology the Hyaluronidase domainof other hyaluronidase-like sequences. The glycoprotein exhibitsHyaluronidase activity as a single chain.

As used herein, by homologous means about greater than 25% nucleic acidsequence identity, such as 25% 40%, 60%, 70%, 80%, 90% or 95%. Ifnecessary the percentage homology will be specified. The terms“homology” and “identity” are often used interchangeably. In general,sequences are aligned so that the highest order match is obtained (see,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; Carrillo et al. (1988) Slam J Applied Math 48:1073-1082).

By sequence identity, the numbers of conserved amino acids is determinedby standard alignment algorithms programs, and are used with default gappenalties established by each supplier. Substantially homologous nucleicacid molecules would hybridize typically at moderate stringency or athigh stringency all along the length of the nucleic acid or along atleast about 70%, 80% or 90% of the full-length nucleic acid molecule ofinterest. Also contemplated are nucleic acid molecules that containdegenerate codons in place of codons in the hybridizing nucleic acidmolecule.

Whether any two nucleic acid molecules have nucleotide sequences thatare at least, for example, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%“identical” can be determined using known computer algorithms such asthe “FASTA” program, using for example, the default parameters as inPearson et al (1988) Proc. Natl. Acad. Sci. USA 85: 2444 (other programsinclude the GCG program package (Devereux, J. et al., Nucleic AcidsResearch 12: 387 (1984)), BLASTP, BLASTN, FASTA (Altschul et al., JMolec Biol 215: 403-410 (1990); Guide to Huge Computers, Martin J.Bishop, Ed., Academic Press, San Diego, 1994, and Carrillo et al. (1988)Slam J Applied Math 48:1073-1082). For example, the BLAST function ofthe National Center for Biotechnology Information database can be usedto determine identity. Other commercially or publicly available programsinclude, DNASTAR″ MEGALIGN″ PROGRAM (Madison, Wis.) and the Universityof Wisconsin Genetics Computer Group (UWG) “Gap” program (MadisonWis.)). Percent homology or identity of proteins and/or nucleic acidmolecules can be determined, for example, by comparing sequenceinformation using a GAP computer program e.g. Needleman et al. (1970), JMol Biol. 48: 443, as revised by Smith and Waterman Adv. Appl. Math(1981) 2:482). Briefly, the GAP program defines similarity as the numberof aligned symbols (i.e., nucleotides or amino acids) that are similar,divided by the total number of symbols in the shorter of the twosequences. Default parameters for the GAP program can include: (1) aunary comparison matrix (containing a value of 1 for identities and 0for non-identities) and the weighted comparison matrix of Gribskov et al(1986) Nucl. Acids Res. 14: 6745, as described by Schwartz and Dayhoff,eds., Atlas Of Protein Sequence And Structure, National BiomedicalResearch Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for eachgap and an additional 0.10 penalty for each symbol in each gap; and (3)no penalty for end gaps. Therefore, as used herein, the term “identity”represents a comparison between a test and a reference polypeptide orpolynucleotide.

As used herein, the term at least “90% identical to” refers to percentidentities from 90 to 99.99 relative to the reference polypeptides.Identity at a level of 90% or more is indicative of the fact that,assuming for exemplification purposes a test and referencepolynucleotide length of 100 amino acids are compared. No more than 10%(i.e., 10 out of 100) amino acids in the test polypeptide differ fromthat of the reference polypeptides. Similar comparisons can be madebetween a test and reference polynucleotides. Such differences can berepresented as point mutations randomly distributed over the entirelength of an amino acid sequence or they can be clustered in one or morelocations of varying length up to the maximum allowable, e.g. 10/100amino acid difference (approximately 90% identity). Differences aredefined as nucleic acid or amino acid substitutions, or deletions. Atthe level of homologies or identities above about 85-90%, the resultshould be independent of the program and gap parameters set; such highlevels of identity can be assessed readily, often without relying onsoftware.

As used herein, primer refers to an oligonucleotide containing two ormore deoxyribonucleotides or ribonucleotides, typically more than three,from which synthesis of a primer extension product can be initiated.Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization and extension,such as DNA polymerase, and a suitable buffer, temperature and pH.

As used herein, animals include any animal, such as, but are not limitedto, goats, cows, deer, sheep, rodents, pigs and humans. Non-humananimals, exclude humans as the contemplated animal. The sHASEGPsprovided herein are from any source, animal, plant, prokaryotic andfungal. Most sHASEGP's are of animal origin, including mammalian origin.

As used herein, genetic therapy involves the transfer of heterologousnucleic acid, such as DNA, into certain cells, target cells, of amammal, particularly a human, with a disorder or conditions for whichsuch therapy is sought. The nucleic acid, such as DNA, is introducedinto the selected target cells in a manner such that the heterologousnucleic acid, such as DNA, is expressed and a therapeutic productencoded thereby is produced.

Alternatively, the heterologous nucleic acid, such as DNA, can in somemanner mediate expression of DNA that encodes the therapeutic product,or it can encode a product, such as a peptide or RNA that in some mannermediates, directly or indirectly, expression of a therapeutic product.Genetic therapy can also be used to deliver nucleic acid encoding a geneproduct that replaces a defective gene or supplements a gene productproduced by the mammal or the cell in which it is introduced. Theintroduced nucleic acid can encode a therapeutic compound, such as agrowth factor inhibitor thereof, or a tumor necrosis factor or inhibitorthereof, such as a receptor therefore, that is not normally produced inthe mammalian host or that is not produced in therapeutically effectiveamounts or at a therapeutically useful time. The heterologous nucleicacid, such as DNA, encoding the therapeutic product can be modifiedprior to introduction into the cells of the afflicted host in order toenhance or otherwise alter the product or expression thereof. Genetictherapy can also involve delivery of an inhibitor or repressor or othermodulator of gene expression.

As used herein, heterologous nucleic acid is nucleic acid that (if DNAencodes RNA) and proteins that are not normally produced in vivo by thecell in which it is expressed or that mediates or encodes mediators thatalter expression of endogenous nucleic acid, such as DNA, by affectingtranscription, translation, or other regulatable biochemical processes.Heterologous nucleic acid, such as DNA, can also be referred to asforeign nucleic acid, such as DNA. Any nucleic acid, such as DNA, thatone of skill in the art would recognize or consider as heterologous orforeign to the cell in which is expressed is herein encompassed byheterologous nucleic acid; heterologous nucleic acid includesexogenously added nucleic acid that is also expressed endogenously.Examples of heterologous nucleic acid include, but are not limited to,nucleic acid that encodes traceable marker proteins, such as a proteinthat confers drug resistance, nucleic acid that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and nucleic acid, such as DNA, that encodes other types of proteins,such as antibodies. Antibodies that are encoded by heterologous nucleicacid can be secreted or expressed on the surface of the cell in whichthe heterologous nucleic acid has been introduced.

Heterologous nucleic acid is generally not endogenous to the cell intowhich it is introduced, but has been obtained from another cell orprepared synthetically.

Generally, although not necessarily, such nucleic acid encodes RNA andproteins that are not normally produced by the cell in which it isexpressed.

As used herein, a therapeutically effective product is a product that isencoded by heterologous nucleic acid, typically DNA, that, uponintroduction of the nucleic acid into a host, a product is expressedthat ameliorates or eliminates the symptoms, manifestations of aninherited or acquired disease or that cures the disease.

As used herein, recitation that a glycoprotein consists essentially ofthe Hyaluronidase domain means that the only sHASEGP portion of thepolypeptide is a Hyaluronidase domain or a catalytically active portionthereof. The polypeptide can optionally, and generally will, includeadditional non-sHASEGP-derived sequences of amino acids.

As used herein, domain refers to a portion of a molecule, e.g.,glycoproteins or the encoding nucleic acids that is structurally and/orfunctionally distinct from other portions of the molecule.

As used herein, Hyaluronidase refers to an enzyme catalyzing hydrolysisof glycosaminoglycans.

For clarity reference to Hyaluronidase refers to all forms, andparticular forms will be specifically designated. For purposes herein,the Hyaluronidase domain includes the membrane bound and soluble formsof a sHASEGP protein.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including protein nucleic acids (PNA) and mixture thereof. Nucleic acidscan be single or double-stranded. When referring to probes or primers,optionally labeled, with a detectable label, such as a fluorescent orradiolabel, single-stranded molecules are contemplated. Such moleculesare typically of a length such that their target is statistically uniqueor of low copy number (typically less than 5, generally less than 3) forprobing or priming a library. Generally a probe or primer contains atleast 14, 16 or 30 contiguous of sequence complementary to or identicala gene of interest. Probes and primers can be 10, 20, 30, 50, 100 ormore nucleic acids long.

As used herein, nucleic acid encoding a fragment or portion of a sHASEGPrefers to a nucleic acid encoding only the recited fragment or portionof sHASEGP, and not the other contiguous portions of the sHASEGP.

As used herein, operative linkage of heterologous nucleic to regulatoryand effector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signal sequencesrefers to the relationship between such nucleic acid, such as DNA, andsuch sequences of nucleotides. For example, operative linkage ofheterologous DNA to a promoter refers to the physical relationshipbetween the DNA and the promoter such that the transcription of such DNAis initiated from the promoter by an RNA polymerase that specificallyrecognizes, binds to and transcribes the DNA in reading frame. Thus,operatively linked or operationally associated refers to the functionalrelationship of nucleic acid, such as DNA, with regulatory and effectorsequences of nucleotides, such as promoters, enhancers, transcriptionaland translational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itcan be necessary to remove, add or alter 5′ Untranslated portions of theclones to eliminate extra, potential inappropriate alternativetranslation initiation i.e. start) codons or other sequences that caninterfere with or reduce expression, either at the level oftranscription or translation. Alternatively, consensus ribosome bindingsites (see, e.g., Kozak J. Biol. Chem. 266: 19867-19870 (1991) can beinserted immediately 5′ of the start codon and can enhance expression.The desirability of (or need for) such modification can be empiricallydetermined.

As used herein, a sequence complementary to at least a portion of anRNA, with reference to antisense oligonucleotides, means a sequencehaving sufficient complimentary to be able to hybridize with the RNA,generally under moderate or high stringency conditions, forming a stableduplex; in the case of double-stranded sHASEGP antisense nucleic acids,a single strand of the duplex DNA (or dsRNA) can thus be tested, ortriplex formation can be assayed. The ability to hybridize depends onthe degree of complimentarity and the length of the antisense nucleicacid. Generally, the longer the hybridizing nucleic acid, the more basemismatches with a sHASEGP encoding RNA it can contain and still form astable duplex (or triplex, as the case can be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

For purposes herein, amino acid substitutions can be made in any ofsHASEGPs and Hyaluronidase domains thereof provided that the resultingprotein exhibits Hyaluronidase activity. Amino acid substitutionscontemplated include conservative substitutions, such as those set forthin Table 1, which do not eliminate proteolytic activity. As describedherein, substitutions that alter properties of the proteins, such asremoval of cleavage sites and other such sites are also contemplated;such substitutions are generally non-conservative, but can be readilyeffected by those of skill in the art.

Suitable conservative substitutions of amino acids are known to those ofskill in this art and can be made generally without altering thebiological activity, for example enzymatic activity, of the resultingmolecule. Those of skill in this art recognize that, in general, singleamino acid substitutions in non-essential regions of a polypeptide donot substantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224). Also included within the definition, is thecatalytically active fragment of a sHASEGP, particularly a single chainHyaluronidase portion. Conservative amino acid substitutions are made,for example, in accordance with those set forth in TABLE 1 as follows:

TABLE 1 Original residue Conservative substitution Ala (A) Gly; Ser, AbuArg (R) Lys, orn Asn (N) Gln; His Cys (C) Ser Gin (O) Asn Glu (E) ASPGly (G) Ala; Pro His (H) Asn; Gin Ile (I) Leu; Val; Met; Nle; Nva Leu(L); Val; Met; Nle; Nv Lys (K) Arg; Gin; Glu Met (M) Leu; Tyr; Ile; NLeVal Ornitine Lys; Arg Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp(W) Tyr Tyr (Y) Trp; Phe Val (V) ILE; Leu; Met; Nle; Nv Othersubstitutions are also permissible and can be determined empirically orin accord with known conservative substitutions.

As used herein, Abu is 2-aminobutyric acid; Orn is ornithine. As usedherein, the amino acids, which occur in the various amino acid sequencesappearing herein, are identified according to their well-known,three-letter or one-letter abbreviations. The nucleotides, which occurin the various DNA fragments, are designated with the standardsingle-letter designations used routinely in the art.

As used herein, a probe of primer based on a nucleotide sequencedisclosed herein, includes at least 10, 14, typically at least 16contiguous sequence of nucleotides of SEQ ID NO. 6, and probes of atleast 30, 50 or 100 contiguous sequence of nucleotides of SEQ ID NO. 6.The length of the probe or primer for unique hybridization is a functionof the complexity of the genome of interest.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient that canbe attributed to or associated with administration of the composition.

As used herein, antisense polynucleotides refer to synthetic sequencesof nucleotide bases complementary to mRNA or the sense strand ofdouble-stranded DNA. Admixture of sense and antisense polynucleotidesunder appropriate conditions leads to the binding of the two molecules,or hybridization. When these polynucleotides bind to (hybridize with)mRNA, inhibition of protein synthesis (translation) occurs. When thesepolynucleotides bind to double-stranded DNA, inhibition of RNA synthesis(transcription) occurs.

The resulting inhibition of translation and/or transcription leads to aninhibition of the synthesis of the protein encoded by the sense strand.Antisense nucleic acid molecule typically contain a sufficient number ofnucleotides to Specifically bind to a target nucleic acid, generally atleast 5 contiguous nucleotides, often at least 14 or 16 or 30 contiguousnucleotides or modified nucleotides complementary to the coding portionof a nucleic acid molecule that encodes a gene of interest, for example,nucleic acid encoding a single chain Hyaluronidase domain of an sHASEGP.

As used herein, an array refers to a collection of elements, such asantibodies, containing three or more members. An addressable array isone in which the members of the array are identifiable, typically byposition on a solid phase support. Hence, in general the members of thearray are immobilized on discrete identifiable loci on the surface of asolid phase.

As used herein, antibody refers to an immunoglobulin, whether natural orpartially or wholly synthetically produced, including any derivativethereof that retains the specific binding ability the antibody. Henceantibody includes any protein having a binding domain that is homologousor substantially homologous to an immunoglobulin-binding domain.Antibodies include members of any immunoglobulin claims, including IgG,IgM, IgA, IgD and IgE.

As used herein, antibody fragment refers to any derivative of anantibody that is less then full-length, retaining at least a portion ofthe full-length antibody's specific binding ability. Examples ofantibody fragments include, but are not limited to Fab, Fab′, F(AB)₂,single chain Fvs (scFV), FV, dsFV diabody and Fd fragments. The fragmentcan include multiple chains linked together, such as by disulfidebridges. An antibody fragment generally contains at least about 50 aminoacids and typically at least 200 amino acids.

As used herein, an Fv antibody fragment is composed of one variableheavy domain (VH) and one variable light domain linked by noncovalentinteractions.

As used herein, a dsFV refers to an Fv with an engineered intermoleculardisulfide bond.

As used herein, an F (AB)2 fragment is an antibody fragment that resultsfrom digestion of an immunoglobulin with pepsin at pH 4.0-4.5; it can berecombinantly expressed to produce the equivalent fragment.

As used herein, Fab fragments are antibody fragments that result fromdigestion of an immunoglobulin with papain; they can be recombinantlyexpressed to produce the equivalent fragment.

As used herein, scFVs refer to antibody fragments that contain avariable light chain V, and variable heavy chain (VH) covalentlyconnected by a polypeptide linker in any order. The linker is of alength such that the two variable domains are bridged withoutsubstantial interference. Included linkers are (Gly-Ser) n residues withsome Glu or Lys residues dispersed throughout to increase solubility.

As used herein, humanized antibodies refer to antibodies that aremodified to include human sequences of amino acids so thatadministration to a human does not provoke an immune response. Methodsfor preparation of such antibodies are known. For example, to producesuch antibodies, the hybridoma or other prokaryotic or eukaryotic cell,such as an E. coli or a CHO cell, that expresses the monoclonal antibodyare altered by recombinant DNA techniques to express an antibody inwhich the amino acid composition of the non-variable region is based onhuman antibodies. Computer programs have been designed to identify suchregions.

As used herein, diabodies are dimeric scFV; diabodies typically haveshorter peptide linkers than ScFVs, and they generally dimerize.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein the term assessing is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of an sHASEGP, or a domain thereof, present inthe sample, and also of obtaining an index, ratio, percentage, visual orother value indicative of the level of the activity. Assessment can bedirect or indirect and the chemical species actually detected need notof course be the proteolysis product itself but can for example be aderivative thereof or some further substance.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein the biological activity of aluciferase is its oxygenase activity whereby, upon oxidation of asubstrate, light is produced.

As used herein, functional activity refers to a polypeptide or portionthereof that displays one or more activities associated with afull-length protein.

Functional activities include, but are not limited to, biologicalactivity, catalytic or enzymatic activity, antigenicity (ability to bindto or compete with a polypeptide for binding to an anti-polypeptideantibody), immunogenicity, ability to form multimers, the ability toSpecifically bind to a receptor or ligand for the polypeptide.

As used herein, a conjugate refers to the compounds provided herein thatincludes one or more sHASEGPs, including a sHASEGP, particularly singlechain Hyaluronidase domains thereof, and one or more targeting agents.These conjugates include those produced by recombinant means as fusionproteins, those produced by chemical means, such as by chemicalcoupling, through, for example, coupling to sulfhydryl groups, and thoseproduced by any other method whereby at least one sHASEGP, or a domainthereof, is linked, directly or indirectly via linker (s) to a targetingagent.

As used herein, a targeting agent is any moiety, such as a protein oreffective portion thereof, that provides specific binding of theconjugate to a cell surface receptor, which, can internalize theconjugate or sHASEGP portion thereof. A targeting agent can also be onethat promotes or facilitates, for example, affinity isolation orpurification of the conjugate; attachment of the conjugate to a surface;or detection of the conjugate or complexes containing the conjugate.

As used herein, an antibody conjugate refers to a conjugate in which thetargeting agent is an antibody.

As used herein, derivative or analog of a molecule refers to a portionderived from or a modified version of the molecule.

As used herein, an effective amount of a compound for treating aparticular disease is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. Such amountcan be administered as a single dosage or can be administered accordingto a regimen, whereby it is effective. The amount can cure the diseasebut, typically, is administered in order to ameliorate the symptoms ofthe disease. Repeated administration can be required to achieve thedesired amelioration of symptoms.

As used herein equivalent, when referring to two sequences of nucleicacids means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with only aminoacid substitutions (such, as but not limited to, conservative changessuch as those set forth in Table 1, above) that do not substantiallyalter the activity or function of the protein or peptide. Whenequivalent refers to a property, the property does not need to bepresent to the same extent (e.g., two peptides can exhibit differentrates of the same type of enzymatic activity), but the activities areusually substantially the same. Complementary, when referring to twonucleotide sequences, means that the two sequences of nucleotides arecapable of hybridizing, typically with less than 25%, 15%, 5% or 0%mismatches between opposed nucleotides. If necessary the percentage ofcomplimentarily will be specified. Typically the two molecules areselected such that they will hybridize under conditions of highstringency.

As used herein, an agent that modulates the activity of a protein orexpression of a gene or nucleic acid either decreases or increases orotherwise alters the activity of the protein or, in some manner up- ordown-regulates or otherwise alters expression of the nucleic acid in acell.

As used herein, inhibitor of the activity of an sHASEGP encompasses anysubstance that prohibits or decrease production, post-translationalmodification (s), maturation, or membrane localization of the sHASEGP orany substance that interferes with or decreases the proteolytic efficacyof thereof, particularly of a single chain form in an in vitro screeningassay.

As used herein, a method for treating or preventing neoplastic diseasemeans that any of the symptoms, such as the tumor, metastasis thereof,the vascularization of the tumors or other parameters by which thedisease is characterized are reduced, ameliorated, prevented, placed ina state of remission, or maintained in a state of remission. It alsomeans that the hallmarks of neoplastic disease and metastasis can beeliminated, reduced or prevented by the treatment. Non-limiting examplesof the hallmarks include uncontrolled degradation of the basementmembrane and proximal extracellular matrix, migration, division, andorganization of the endothelial cells into new functioning capillaries,and the persistence of such functioning capillaries.

As used herein, pharmaceutically acceptable salts, esters or otherderivatives of the conjugates include any salts, esters or derivativesthat can be readily prepared by those of skill in this art using knownmethods for such derivatization and that produce compounds that can beadministered to animals or humans without substantial toxic effects andthat either are pharmaceutical active or are prodrugs.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutical active compound ismodified such that the active compound is regenerated by metabolicprocesses. The prodrug can be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388-392).

As used herein, a drug identified by the screening methods providedherein refers to any compound that is a candidate for use as atherapeutic or as a lead compound for the design of a therapeutic. Suchcompounds can be small molecules, including small organic molecules,peptides, peptide mimetics, antisense molecules or dsRNA, such as RNAi,antibodies, fragments of antibodies, recombinant antibodies and othersuch compounds that can serve as drug candidates or lead compounds.

As used herein, a peptidomimetic is a compound that mimics theconformation and certain stereochemical features of the biologicallyactive form of a particular peptide. In general, peptidomimetics aredesigned to mimic certain desirable properties of a compound, but notthe undesirable properties, such as flexibility, that lead to a loss ofa biologically active conformation and bond breakdown. Peptidomimeticsmay be prepared from biologically active compounds by replacing certaingroups or bonds that contribute to the undesirable properties withbioisosteres. Bioisosteres are known to those of skill in the art. Forexample the methylene bioisostere CH2S has been used as an amidereplacement in enkephalin analogs (see, e.g. Spatola (1983) pp. 267-357in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins,Weistein, Ed. volume 7, Marcel Dekker, New York). Morphine, which can beadministered orally, is a compound that is a peptidomimetic of thepeptide endorphin. For purposes herein, cyclic peptides are includedamong pepidomimetics.

As used herein, a promoter region or promoter element refers to asegment of DNA or RNA that controls transcription of the DNA or RNA towhich it is operatively linked. The promoter region includes specificsequences that are sufficient for RNA polymerase recognition, bindingand transcription initiation.

This portion of the promoter region is referred to as the promoter. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences can be cis acting or can be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, can be constitutive or regulated. Exemplary promoterscontemplated for use in prokaryotes include the bacteriophage T7 and T3promoters.

As used herein, a receptor refers to a molecule that has an affinity fora given ligand. Receptors can be naturally occurring or syntheticmolecules. Receptors can also be referred to in the art as anti-ligands.As used herein, the receptor and anti-ligand are interchangeable.Receptors can be used in their unaltered state or as aggregates withother species. Receptors can be attached, covalently or noncovalently,or in physical contact with, to a binding member, either directly orindirectly via a specific binding substance or linker. Examples ofreceptors, include, but are not limited to: antibodies, cell membranereceptors surface receptors and internalizing receptors, monoclonalantibodies and antisera reactive with specific antigenic determinantssuch as on viruses, cells, or other materials], drugs, polynucleotides,nucleic acids, peptides, factors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles.

Examples of receptors and applications using such receptors, include butare not restricted to: a) enzymes: specific transport proteins orenzymes essential to survival of microorganisms, which could serve astargets for antibiotic [ligand] selection; b) antibodies: identificationof a ligand-binding site on the antibody molecule that combines with theepitope of an antigen of interest can be investigated; determination ofa sequence that mimics an antigenic epitope can lead to the developmentof vaccines of which the immunogen is based on one or more of suchsequences or lead to the development of related diagnostic agents orcompounds useful in therapeutic treatments such as for auto-immunediseases c) nucleic acids: identification of ligand, such as protein orRNA, binding sites; d) catalytic polypeptides: polymers, includingpolypeptides, that are capable of promoting a chemical reactioninvolving the conversion of one or more reactants to one or moreproducts; such polypeptides generally include a binding site specificfor at least one reactant or reaction intermediate and an activefunctionality proximate to the binding site, in which the functionalityis capable of chemically modifying the bound reactant (see, e.g., U.S.Pat. No. 5,215,899); e) hormone receptors: determination of the ligandsthat bind with high affinity to a receptor is useful in the developmentof hormone replacement therapies; for example, identification of ligandsthat bind to such receptors can lead to the development of drugs tocontrol blood pressure; and f) opiate receptors: determination ofligands that bind to the opiate receptors in the brain is useful in thedevelopment of less-addictive replacements for morphine and relateddrugs.

As used herein, sample refers to anything that can contain an analytefor which an analyte assay is desired. The sample can be a biologicalsample, such as a biological fluid or a biological tissue. Examples ofbiological fluids include urine, blood, plasma, serum, saliva, semen,stool, sputum, cerebral spinal fluid, tears, mucus, sperm, amnioticfluid or the like. Biological tissues are aggregate of cells, usually ofa particular kind together with their intercellular substance that formone of the structural materials of a human, animal, plant, bacterial,fungal or viral structure, including connective, epithelium, muscle andnerve tissues. Examples of biological tissues also include organs,tumors, lymph nodes, arteries and individual cells.

As used herein: stringency of hybridization in determining percentagemismatch is as follows: 1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.2) medium stringency: 0.2×SspE, 0.1% SDS, 50° C. 3 low stringency:1.0×SspE, 0.1% SDS, 50° C. Those of skill in this art know that thewashing step selects for stable hybrids and also know the ingredients ofSspE (see, e.g., Sambrook, E. F. Fritsch, T. Maniatis, in: MolecularCloning, A Laboratory Manual, Cold spring Harbor Laboratory Press 1989Vol 3, p. B. 13, see, also, numerous catalogs that describe commonlyused laboratory solutions). SspE is pH 7.4 phosphate-buffered 0.18 NaCl.Further, those of skill in the art recognize that the stability ofhybrids is determined by TmT which is a function of the sodium ionconcentration and temperature (T_(m)=81.5° C.−16.6+0.41 (% G+C)−600/L))so that the only parameters in the wash conditions critical to hybridstability are sodium ion concentration in the SspE (or SSC) andtemperature.

It is understood that equivalent stringencies can be achieved usingalternative buffers, salts and temperatures. By way of example and notlimitation, procedures using conditions of low stringency are as follows(see also Shilo and Weinberg, Proc. Natl. Acad Sci USA 78: 6789-6792(1981)): Filters containing DNA are pretreated for 6 hours at 40 C in asolution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.1% PVP, 0.1% Ficoll 1% BSA, and 500 ug/ml Denatured Salmon spermDNA (10×) SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate,adjusted to a pH of 7).

Hybridizations are carried out in the same solution with the followingmodifications: 0.02% PVP, 0.02% Ficoll 0.2% BSA, 100VG/M sperm DNA, 10%(wt/vol) dextran sulfate, and 5-20×106 cpm 32P-labeled probe is used.Filters are incubated in hybridization mixture for 18-20 hours at 40 Cand then washed for 1.5 hours at 55 C in a solution containing 2×SSC, 25mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 hours at 60C. Filters are blotted dry and exposed for autoradiography. Ifnecessary, filters are washed for a third time at 65-68 C and reexposedto film. Other conditions of low stringency which can be used are wellknown in the art e.g. as employed for cross-species hybridizations).

By way of example and not way of limitation, procedures using conditionsof moderate stringency include, for example, but are not limited to,procedures using such conditions of moderate stringency are as follows:Filters containing DNA are pretreated for 6 hours at 55 C in a solutioncontaining 6×SSC, 5×Denhart's solution, 0.5% SDS and 100 ug/ml denaturedsalmon sperm DNA. Hybridizations are carried out in the same solutionand 5-20×106 32P labeled probe is used. Filters are incubated inhybridization mixture for 18-20 hours at 55 C and then washed twice for30 minutes at 60 C in a solution containing 1×SSC and 0.1% SDS. Filtersare blotted dry and exposed for autoradiography. Other conditions ofmoderate stringency that can be used are well known in the art. Washingof filters is done at 37 C for 1 hour in a solution containing 2×SSC,0.1% SDS.

By way of example and not way of limitation, procedures using conditionsof high stringency are as follows: Prehybridization of filterscontaining DNA is carried out for 8 hours to overnight at 65 C in buffercomposed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%Ficoll, 0.02% BSA, and 500 ug/ml denatured salmon sperm DNA. Filters arehybridized for 48 hours at 65° C. in prehybridization mixture containing100 ug/ml denatured salmon sperm DNA and 5-20×106 CPM 32P labeled probe.Washing of filters is done at 37 C for 1 hour in a solution containing2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by awash in 0.1×SSC at 50 C for 45 minutes before autoradiography. Otherconditions of high stringency that can be used are well known in theart.

The term substantially identical or substantially homologous or similarvaries with the context as understood by those skilled in the relevantart and generally means at least 60% or 70%, preferably means at least80%, 85% or more preferably at least 90%, and most preferably at least95% identity.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound can, however, be amixture of stereoisomers or isomers. In such instances, furtherpurification might increase the specific activity of the compound.

As used herein, target cell refers to a cell that expresses a sHASEGP invivo.

As used herein, test substance (or test compound) refers to a chemicallydefined compound (e.g., organic molecules, inorganic molecules,organic/inorganic molecules, proteins, peptides, nucleic acids,oligonucleotides, lipids, polysaccharides, saccharides, or hybrids amongthese molecules such as glycoproteins, etc.) or mixtures of compounds(e.g., a library of test compounds, natural extracts or culturesupernatants, etc.) whose effect on an sHASEGP, particularly a singlechain form that includes the Hyaluronidase domain or a sufficientportion thereof for activity, as determined by an in vitro method, suchas the assays provided herein.

As used herein, the terms a therapeutic agent, therapeutic regimen,radioprotectant or chemotherapeutic mean conventional drugs and drugtherapies, including vaccines, which are known to those skilled in theart. Radiotherapeutic agents are well known in the art.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered.

Treatment also encompasses any pharmaceutical use of the compositionsherein.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vehiclesare well known to those of skill in the art. An expression vectorincludes vectors capable of expressing DNA that is operatively linkedwith regulatory sequences, such as promoter regions, that are capable ofeffecting expression of such DNA fragments. Thus, an expression vectorrefers to a recombinant DNA or RNA construct, such as a plasmid, aphage, recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, protein binding sequence refers to a protein or peptidesequence that is capable of specific binding to other protein or peptidesequences generally, to a set of protein or peptide sequences or to aparticular protein or peptide sequence.

As used herein, epitope tag refers to a short stretch of amino acidresidues corresponding to an epitope to facilitate subsequentbiochemical and immunological analysis of the epitope tagged protein orpeptide. Epitope tagging is achieved by including the sequence of theepitope tag to the protein-encoding sequence in an appropriateexpression vector. Epitope tagged proteins can be affinity purifiedusing highly specific antibodies raised against the tags.

As used herein, metal binding sequence refers to a protein or peptidesequence that is capable of specific binding to metal ions generally, toa set of metal ions or to a particular metal ion.

As used herein, a combination refers to any association between two oramong more items.

As used herein, a composition refers to any mixture. It can be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a cellular extract refers to a preparation or fractionwhich is made from a lysed or disrupted cell.

As used herein, an agent is said to be randomly selected when the agentis chosen randomly without considering the specific sequences involvedin the association of a protein alone or with its associated substrates,binding partners, etc. An example of randomly selected agents is the usea chemical library or a peptide combinatorial library, or a growth brothof an organism or conditioned medium.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a non-random basis that takes into accountthe sequence of the target site and/or its conformation in connectionwith the agents action. As described in the Examples, there are proposedbinding sites for Hyaluronidase and (catalytic) sites in theglycoprotein having SEQ ID NO: 1 or SEQ ID NO: 4. Agents can berationally selected or rationally designed by utilizing the peptidesequences that make up these sites. For example, a rationally selectedpeptide agent can be a peptide whose amino acid sequence is identical tothe ATP or calmodulin binding sites or domains.

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right. All oligosaccharides describedherein are described with the name or abbreviation for the non-reducingsaccharide (e.g., Gal), followed by the configuration of the glycosidicbond (.alpha. or. beta.), the ring bond, the ring position of thereducing saccharide involved in the bond, and then the name orabbreviation of the reducing saccharide (e.g., GlcNAc). The linkagebetween two sugars may be expressed, for example, as 2,3, 2.fw darw.3,or (2,3). Each saccharide is a pyranose.

As used herein, N-linked sugar moiety refers to an oligosaccharideattached to a sHASEGP via the amide nitrogen of Asn residues. N-linkedoligosaccharides fall into several major types (oligomannose, complex,hybrid, sulfated), all of which have (Man) 3-GlcNAc-GlcNAc-coresattached via the amide nitrogen of Asn residues that fall within-Asn-Xaa-Thr/Ser-sequences (where Xaa is not Pro). N-linked sites areoften indirectly assigned by the appearance of a “blank” cycle duringsequencing. Positive identification can be made after release of theoligosaccharide by PNGase F, which converts the glycosylated Asn to Asp.After PNGase F release, N-linked oligosaccharides can be purified usingBio-Gel P-6 chromatography, with the oligosaccharide pool subjected topreparative high pH anion exchange chromatography (HPAEC) (Townsend etal., (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide isomerscan be resolved using HPAEC. Fucose residues will shift elutionpositions earlier in the HPAEC chromatogram, while additional sialicacid residues will increase the retention time. Concurrent treatment ofglycoproteins whose oligosaccharide structures are known (e.g., bovinefetuin, a-l acid glycoprotein, ovalbumin, RNAse B, transferrin) canfacilitate assignment of the oligosaccharide peaks. The collectedoligosaccharides can be characterized by a combination of compositionaland methylation linkage analyses (Waegheet al., (1983) Carbohydr Res.123, 281-304.), with anomeric configurations assigned by NMRspectroscopy (Van Halbeek (1993) in Methods Enzymol 230).

Alternatively, oligosaccharides can be identified by fluorescenceassisted carbohydrate electrophoresis (FACE) Callewaert et al. (2001)Glycobiology 11, 275-281.

As used herein, the term “sialic acid” refers to any member of a familyof nine-carbon carboxylated sugars. The most common member of the sialicacid family is N-acetylneuraminic acid(2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al. (1990) J. Biol. Chem. 265:21811-21819. Also included are 9-substituted sialic acids such as a9-O—C.sub.1-C.sub.6 acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac.For review of the sialic acid family, see, e.g., Varki (1992)Glycobiology 2: 25-40; Sialic Acids: Chemistry, Metabolism and Function,R. Schauer, Ed. (Springer-Verlag, N.Y. (1992)). The synthesis and use ofsialic acid compounds in a sialation procedure is disclosed ininternational application WO 92/16640, published Oct. 1, 1992.

As used herein, PNGase refers to an Asparagine Peptide specificN-glycosidase F such as the Flavobacterium maningoseptumpeptide-N-glycosidase F. PNGASE enzymes are characterized by theirspecificity towards N-linked rather than O-linked oligosaccharides.Characterization of PNGASE efficacy can be defined by both SDS PAGEelectrophoresis, or fluorescent assisted carbohydrate electrophoresis.

As used herein substantially terminated Sialation refers to N-linkedoligosaccharides terminating with sialic acid residue as a terminalsugar. Terminal sialic acids can be identified by FACE analysis ofreleased carbohydrates following treatment with neuraminidase.

The circulatory lifetime of glycoproteins in the blood is highlydependent on the composition and structure of its N-linked carbohydrategroups. This fact is of direct relevance for therapeutic glycoproteinsthat are intended to be administered parenterally. In general, maximalcirculatory half-life of a glycoprotein requires that its N-linkedcarbohydrate groups terminate in the sequence NeuAc-Gal-GlcNAc. Withoutthe terminal sialic acid (NeuAc), the glycoprotein is rapidly clearedfrom the blood by a mechanism involving the recognition of theunderlying N-acetylgalactosamine (GalNAc) or galactose (Gal) residues(Goochee et al. (1991) Biol/Technology 9: 1347-1355). For this reason,ensuring the presence of terminal sialic acid on N-linked carbohydrategroups of therapeutic glycoproteins is an important consideration fortheir commercial development.

Circulating glycoproteins are exposed to sialidase(s) (or neuraminidase)which can remove terminal sialic acid residues. Typically the removal ofthe sialic acid exposes galactose residues, and these residues arerecognized and bound by galactose-specific receptors in hepatocytes(reviewed in Ashwell and Harford (1982) Ann. Rev. Biochem. 51:531).Liver also contains other sugar-specific receptors which mediate removalof glycoproteins from circulation. Specificities of such receptors alsoinclude N-acetylglucosamine, mannose, fucose and phosphomannose.Glycoproteins cleared by the galactose receptors of hepatocytes undergosubstantial degradation and then enter the bile; glycoproteins clearedby the mannose receptor of Kupffer cells enter the reticuloendothelialsystem (reviewed in Ashwell and Harford (1982) Ann. Rev. Biochem.51:53).

As used herein Neutral Active refers to a sHASEGP glycoprotein withcatalytic activity towards a glycosaminoglycan substrate in vitro at aPH between 5 and 8 under conditions of salt less than 150 mM andbuffering strength less than 50 mM.

As used herein, a stabilized solution refers to a sHASEGP that retainsgreater than 60% of its initial activity after storage at roomtemperature for 30 days.

As used herein unless otherwise specified, a unit is expressed inturbidity reducing units (TRU). One TRU is defined as the amount ofhyaluronidase activity required to reduce the turbidity of an acidifiedsolution of hyaluronic acid and is equivalent to the U.S.P./NationalFormulary (NF XIII) units (NFU). The ELISA-like enzyme assay describedherein can be related to the TRU, the NFU, and U.S.P. unit through astandard curve of a sample of hyaluronidase (e.g., USP or WHO standard)standardized through the U.S.P. Therefore, the enzyme activitiesdetermined by the ELISA-like enzyme assay are actually relative TRU,since enzyme activity is not actually measured using the turbidometricassay (Dorfman et al., 1948, J. Biol. Chem. 172:367).

As used herein, potency is defined by the amount of sHASEGP proteinrequired to degrade substrate in vitro based upon a Turbidity ReducingUnit or Relative Turbidity Reducing Unit.

As used herein, specific activity refers to Units of activity per mgprotein. The amount of sHASEGP protein is defined by the absorption of asolution of sHASEGP at 280 nm assuming a molar extinction coefficient ofapproximately 1.7, in units of M⁻¹ cm⁻¹.

Polyethylene glycol (PEG) has been widely used in biomaterials,biotechnology and medicine primarily because PEG is a biocompatible,nontoxic, nonimmunogenic and water-soluble polymer (Zhao and Harris, ACSSymposium Series 680: 458-72, 1997). In the area of drug delivery, PEGderivatives have been widely used in covalent attachment (i.e.,“PEGylation”) to proteins to reduce immunogenicity, proteolysis andkidney clearance and to enhance solubility (Zalipsky, Adv. Drug Del.Rev. 16:157-82, 1995). Similarly, PEG has been attached to low molecularweight, relatively hydrophobic drugs to enhance solubility, reducetoxicity and alter biodistribution. Typically, PEGylated drugs areinjected as solutions.

A closely related application is synthesis of crosslinked degradable PEGnetworks or formulations for use in drug delivery since much of the samechemistry used in design of degradable, soluble drug carriers can alsobe used in design of degradable gels (Sawhney et al., Macromolecules 26:581-87, 1993). It is also known that intermacromolecular complexes canbe formed by mixing solutions of two complementary polymers. Suchcomplexes are generally stabilized by electrostatic interactions(polyanion-polycation) and/or hydrogen bonds (polyacid-polybase) betweenthe polymers involved, and/or by hydrophobic interactions between thepolymers in an aqueous surrounding (Krupers et al., Eur. Polym J.32:785-790, 1996). For example, mixing solutions of polyacrylic acid(PAAc) and polyethylene oxide (PEO) under the proper conditions resultsin the formation of complexes based mostly on hydrogen bonding.Dissociation of these complexes at physiologic conditions has been usedfor delivery of free drugs (i.e., non-PEGylated). In addition, complexesof complementary polymers have been formed from both homopolymers andcopolymers.

In one aspect, the polyethylene glycol has a molecular weight rangingfrom about 3 kD to about 50 kD, and preferably from about 5 kD to about30 kD. Covalent attachment of the PEG to the drug (known as“PEGylation”) may be accomplished by known chemical synthesistechniques. For example, in one aspect of the present invention, thePEGylation of protein may be accomplished by reacting NHS-activated PEGwith the protein under suitable reaction conditions.

While numerous reactions have been described for PEGylation, those thatare most generally applicable confer directionality; utilise mildreaction conditions, and do not necessitate extensive downstreamprocessing to remove toxic catalysts or bi-products. For instance,monomethoxyPEG (mPEG) has only one reactive terminal hydroxyl, and thusits use limits some of the heterogeneity of the resulting PEG-proteinproduct mixture. Activation of the hydroxyl group at the end of thepolymer opposite to the terminal methoxy group is generally necessary toaccomplish efficient protein PEGylation, with the aim being to make thederivatised PEG more susceptible to nucleophilic attack. The attackingnucleophile is usually the epsilon-amino group of a lysyl residue, butother amines can also react (e.g. the N-terminal alpha-amine or the ringamines of histidine) if local conditions are favorable. A more directedattachment is possible in proteins containing a single lysine orcysteine. The latter residue can be targeted by PEG-maleimide forthiol-specific modification. Alternatively, PEG hydrazide can be reactedwith periodate oxidized sHASEGP and reduced in the presence of NaCNBH₃.More specifically, PEGylated CMP sugars can be reacted with sHASEGP inthe presence of appropriate glycosyl-transferases. One technique is the“PEGylation” technique where a number of polymeric molecules are coupledto the polypeptide in question. When using this technique the immunesystem has difficulties in recognizing the epitopes on the polypeptide'ssurface responsible for the formation of antibodies, thereby reducingthe immune response. For polypeptides introduced directly into thecirculatory system of the human body to give a particular physiologicaleffect (i.e. pharmaceuticals) the typical potential immune response isan IgG and/or IgM response, while polypeptides which are inhaled throughthe respiratory system (i.e. industrial polypeptide) potentially maycause an IgE response (i.e. allergic response). One of the theoriesexplaining the reduced immune response is that the polymeric molecule(s)shield(s) epitope(s) on the surface of the polypeptide responsible forthe immune response leading to antibody formation. Another theory or atleast a partial factor is that the heavier the conjugate is, the morereduced immune response is obtained.

The polymeric molecules coupled to the polypeptide may be any suitablepolymeric molecule with a molecular weight as defined according to theinvention, including natural and synthetic homopolymers, such as polyols(i.e. poly-OH), polyamines (i.e. poly-NH.sub.2) and polycarboxyl acids(i.e. poly-COOH), and further heteropolymers i.e. polymers comprisingone or more different coupling groups e.g. a hydroxyl group and aminegroups.

Examples of suitable polymeric molecules include polymeric moleculesselected from the group comprising polyalkylene oxides (PAO), such aspolyalkylene glycols (PAG), including polypropylene glycols (PEG),methoxypolyethylene glycols (mPEG) and polypropylene glycols,PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG)branched polyethelene glycols (PEGs), polyvinyl alcohol) PVA),polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids,polyethylene-co-maleic acid anhydride, polystyrene-co-malic acidarhydride, dextrans including carboxymethyl-dextrans, heparin,homologous albumin, celluloses, including methylcellulose,carboxymethylcellulose, ethylcellulosia, hydroxyethylcellulosecarboxyethylcellulose and hydroxypropylcellulose, hydrolysates ofchitosan, starches such as hydroxyethyl-starches and hydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guar gum,pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acidhydrolysates and bio-polymers.

Preferred polymeric molecules are non-toxic polymeric molecules such as(m) polyethylene glycol (mPEG) which further requires a relativelysimple chemistry for its covalent coupling to attachment groups on theenzyme's surface.

Generally seen polyalkylene oxides (PAO), such as polyethylene oxides,such as PEG and especially mPEG, are the preferred polymeric molecules,as these polymeric molecules, in comparison to polysaccharides such asdextran, pullulan and the like, have few reactive groups capable ofcross-linking, which is undesirable.

B. Tissue Expression Profiles sHASEGP

While previously thought to be testis specific, human sHASEGP isexpressed in multiple tissues in humans when using more sensitivetechniques such as RT-PCR. The sHASEGP transcript is found in medulla(brain), microvascular endothelium, prostate, breast, retina, pooledhuman melanocyte, fetal heart, and pregnant uterus. sHASEGP is alsoexpressed in germ cell tumors. RT-PCR based detection of sHASEGPtranscripts is generally required to detect levels in tissues other thantestis.

C. Assays for sHASEGP Enzyme Activity

Turbidometric Microtiter Assay for Hyaluronidase Activity

Hyaluronidase activity can be detected by way of a modifiedturbidometric assay in acidified serum solution. The reagents requiredare as follows:

LTV sterilized 2X-deionized water or Braun R5000-01 sterile water forirrigation Hylumed Medical - Sodium Genzyme Advanced 4876 Hyaluronate,High Molecular Biomaterials Weight HA Hyaluronidase Reference StandardUSP 31200  Potassium Acetate, Granular, J T Baker 2914-01 USP, ACSAcetic Acid, Glacial, 99+% Sigma A-6283 Sodium Phosphate MonobasicMallinkrodt 7774 Monohydrate, USP Granular Sodium Phosphate DibasicMallinkrodt 7771 Anhydrous, USP Sodium Chloride, Crystals, GR, EMScienceSX0420-5 ACS Gelatin Hydrolysate Enzymatic Sigma G-0262 Horse Serum,Donor Herd, cell Sigma H-1270 culture tested, Hybridoma culture tested,USA Origin Human Serum Albumin 20% Griffols Hydrochloric Acid, ACSReagent Sigma H-7020 Calcium Chloride, Dihydrate, J T Baker 1336-01Granular, USP, -FCC

The following reagents are prepared: Acetate Buffer Solution—14.0 g ofpotassium acetate and 25.0 mL of glacial acetic acid in water to make1000 mL. Phosphate Buffer Solution—2.5 g of sodium phosphate monobasic,1.0 g of anhydrous sodium phosphate dibasic, and 8.2 g of sodiumchloride in water to make 1000 mL. Enzyme Diluent Stock Solution—500 mLof Phosphate Buffer Solution with 500 mL of water. Enzyme DiluentWorking Solution—33 mg of hydrolyzed gelatin in 50 mL of Enzyme DiluentStock Solution-prepared within 2 hours of use. Sample StabilizationBuffer Solution (“SSB” Soln.)—125 uL of a 20% Human Serum AlbuminSolution and 50 uL of a 1 M Calcium Chloride solution in 50 mL of EnzymeDiluent Working Solution, and mix thoroughly. Serum StockSolution—Dilute 1 volume of Horse Serum with 9 volumes of Acetate BufferSolution. Adjust with 4 N hydrochloric acid to a pH of 3.1 and allow thesolution to stand at room temperature for 18 to 24 hrs. Store thesolution at 4° C., and use within 30 days. Serum Working Solution—10 mLof the Serum Stock Solution in 30 mL of the Acetate Buffer Solution,adjusted to room temperature. Hyaluronic Acid Stock Solution—SodiumHyaluronic Acid to a concentration 5.0 mg/mL in water. Hyaluronic AcidWorking Solution—0.75 mL of the Hyaluronic Acid Stock Solution in 4.25mL of the Phosphate Buffer Solution. Standard Stock Solution—Onecontainer of USP Reference Standard Hyaluronidase to a concentration1000 Units/mL in water, aliquoted into 50 uL portions, and stored at−20° C. Standard Working Solution—40 uL of Standard Stock Solution in960 uL of cold Enzyme Diluent Working Solution to obtain a solutionhaving a known concentration of 40 Units/mL, prepared immediately beforeuse in the assay.

All enzyme samples are diluted in a “Low Protein Binding” 96-well plateaccording to the following guidelines:

a) The range of maximum sensitivity of this assay is between 10-30Units/mL. To minimize the number of times an assay must be repeated inorder to get results that are within range, first determine theapproximate number of total units/mL for the sample, and then choose a(whole number) dilution such that the final concentration isapproximately 20 Units/ml.

b) Minimum Sample volumes needed to perform assay are as follows: FPLCFractions=50 uL, Tissue Culture Supernatants=1 mL,Purified/Concentrated/Final Step Material=10 uL.

c) For samples with serial dilutions, 1:10 dilutions in the “Low ProteinBinding” 96-well plate are made in triplicate by pipetting 360 uL of the“SSB” Solution and 40 uL of Sample into each well.

For preparation of USP Standard prepare the USP Standard Curve in the“Low Protein Binding” 96-well plate as follows:

USP Standard Curve: Enzyme Standard Final Diluent Working Conc.(inWells: Standard: Soln.(in uL): Soln.(in uL): Units/mL): A1-A3 St01 0 10040 B1-B3 St02 20 80 32 C1-C3 St03 40 60 24 D1-D3 St04 60 40 16 E1-E3St05 80 20 8 F1-F3 St06 90 10 4 G1-G3 St07 100 0 0

For preparation of the Hyaluronic Acid Control in columns 1-3, preparethe H.A. Control in the “Flat Bottom” 96-well plate is prepared asfollows:

H.A. Controls: Hyaluronic Acid Enzyme Diluent Wells: Control: WorkingSoln.(in uL): Working Soln.(in uL): H1-H3 Co01 0 60

The Reaction Plate: 30 uL per well of Hyaluronic Acid Working Solutionis pipetted using a 50 uL 8-channel transfer pipette into a “FlatBottom” 96-well microtiter plate leaving wells H1-H3 empty. 60 uL/wellof Enzyme Diluent Working Solution is pipetted into wells H1-H3 of thesame plate as the HA control.

Serum Working Solution: 40 mL of Serum Working Solution is dispensedinto a transfer basin and next to the Heat Block.

Pre-warming stage: Once both plates have been prepared, the Low ProteinBinding 96-Well plate containing the diluted samples, standards,controls and the Flat Bottom 96-well plate containing the HyaluronicAcid Working Solution are placed onto a heat block and allow them towarm for 5 min. at 37° C.

The Reaction is initiated by the addition of Enzyme to Substrate: 30 uLfrom the enzyme plate into all of the wells in column #1 of the 96-Wellflat bottom plate (containing the substrate) using a 5-50 uL 8-channelpipette. The Enzyme/Substrate reaction mixture is aspirated 5 times(drawing the solution up and down with the transfer during the first 15seconds to ensure complete sample mixing. After mixing the enzyme andsubstrate, the tips are ejected and a new set of tips loaded on thetransfer pipettor for the next column. A timer is restarted, and at time(0=0:30, this process is repeated for column 2. At the next 30 secondinterval (t)=1:00, this is repeated process for column 3. This processis repeated moving from left to right across the plate, every 30 secondsuntil all of the wells contain both enzyme and substrate.

Stopping the reaction: When timer reaches 6 minutes (t)=6:00, 240 uL ofthe Serum Working Solution is pippetted into each well, using a 50-300uL 8-channel transfer pipette, into column 1 of the 96-well flat bottomplate from the adjacent 50 mL Reagent Reservoir. The mixture isaspirated 3 times (drawing the solution up and down with the transferPipettor) during the first 10 seconds to ensure complete mixing. Theprocess is repeated every 30 seconds, proceeding from column's 1 to 12.Upon completion of the last column (column 12), the reaction plate isremoved from the heat block and place the plate onto the read tray ofthe plate reader at 640 nM. A linear curve fit is generated from thestandard curve that permits extrapolation of test samples.

Alternative Assays for Hyaluronidase

Biotinylated Hyaluronan Microtiter Assay

The free carboxyl groups on glucuronic acid residues of Hyaluronan arebiotinylated in a one step reaction using biotin-hydrazide (Pierce),Sulfo NHS (Pierce) and 1-Ethyl dimethylaminopropyl-carbodiimide (Sigma).This biotinylated HA substrate is covalently coupled to a 96 wellmicrotiter plate in a second reaction. At the completion of the enzymereaction, residual substrate is detected with an avidin-peroxidasereaction that can be read in a standard ELISA plate reader. As thesubstrate is covalently bound to the microtiter plate, artifacts such aspH-dependent displacement of the biotinylated substrate does not occur.The sensitivity permits rapid measurement of Hyaluronidase activity fromcultured cells and biological samples with an inter-assay variation ofless than 10%.

The specific activity of hyaluronidase is expressed in turbidityreducing units (TRU). One TRU is defined as the amount of hyaluronidaseactivity required to reduce the turbidity of an acidified solution ofhyaluronic acid and is equivalent to the U.S.P./National Formulary (NFXIII) units (NFU). The ELISA-like enzyme assay used for purification isrelated to the TRU, the NFU, and U.S.P. unit through a standard curve ofa sample of hyaluronidase (e.g., USP) standardized through the U.S.P.Therefore, the enzyme activities determined by the ELISA-like enzymeassay are actually relative TRU, since enzyme activity is not actuallymeasured using the turbidometric assay (Dorfman et al., 1948, J. Biol.Chem. 172:367).

Many Hyaluronidase assays have been based upon the measurement of thegeneration of new reducing N-acetylamino groups (Bonner and Cantey,Clin. Chim. Acta 13:746-752, 1966), or loss of viscosity (De Salegui etal., Arch. Biochem. Biophys. 121:548-554, 1967) or turbidity (Dorfmanand Ott, J. Biol. Chem. 172:367, 1948). With purified substrates all ofthese methods suffice for determination of the presence or absence ofendoglucosamidic activity.

Substantially purified glycosaminoglycan substrates can also be used forin a Gel Shift Assay. Glycosaminoglycans are mixed with recombinantsHASEGP to test for endoglucosidase activity that results in a shift insubstrate mobility within the gel. Chondroitin-4 and 6 sulfate, dermatansulfate, heparan-sulfate can be obtained from Sigma Chemical. Humanumbilical cord Hyaluronan can be obtained from ICN. Each test substrateis diluted to 0.1 mg/ml in a buffer range from pH 3.5-7.5. 10 ul samplesof purified sHASEGP or conditioned media from sHASEGP expressing cellsas well as are mixed with 90 ul of test substrate in desired buffer andincubated for 3 hours at 37 C. Following incubation samples areneutralized with sample buffer (Tris EDTA PH 8.0, Bromophenol Blue andglycerol) followed by electrophoresis. Glycosaminoglycans are detectedby staining the gels in 0.5% Alcian Blue in 3% Glacial Acetic Acidovernight followed by destaining in 7% Glacial Acetic Acid. Degradationis determined by comparison substrate mobility in the presence andabsence of enzyme.

Hyaluronidase activity can also be detected by substrate gel zymography(Guentenhoner et al., 1992, Matrix 388-396). In this assay a sample isapplied to a SDS-PAGE gel containing hyaluronic acid and the proteins inthe sample separated by electrophoresis. The gel is then incubated in anenzyme assay buffer and subsequently stained to detect the hyaluronicacid in the gel. Hyaluronidase activity is visualized as a cleared zonein the substrate gel.

D. Identification and Isolation of sHASEGP Polypeptide Genes

The sHASEGP polypeptide gene and/or domains thereof, can be obtained bymethods well known in the art for DNA isolation. Any method known tothose of skill in the art for identification of nucleic acids thatencode desired genes can be used. Any method available in the art can beused to obtain a full-length (i.e., encompassing the entire codingregion) cDNA or genomic DNA clone encoding a sHASEGP polypeptide. Forexample, the polymerase chain reaction (PCR) can be used to amplify asequence that is expressed in normal tissues, e.g., nucleic acidsencoding a sHASEGP polypeptide (SEQ. Nos: 1 and 2), in a genomic or cDNAlibrary. Oligonucleotide primers that hybridize to sequences at the 3′and 5′ termini of the identified sequences can be used as primers toamplify by PCR sequences from a nucleic acid sample (RNA or DNAgenerally a cDNA library, from an appropriate source (e. testis,prostate, breast).

PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermalcycler and Taq polymerase (Gene Amp). The DNA being amplified caninclude mRNA or cDNA or genomic DNA from any eukaryotic species. One canchoose to synthesize several different degenerate primers, for use inthe PCR reactions.

It is also possible to vary the stringency of hybridization conditionsused in priming the PCR reactions, to amplify nucleic acid homologs(e.g., to obtain sHASEGP polypeptide sequences from species other thanhumans or to obtain human sequences with homology to sHASEGPpolypeptide) by allowing for greater or lesser degrees of nucleotidesequence similarity between the known nucleotide sequence and thenucleic acid homolog being isolated. For cross-species hybridization,low stringency to moderate stringency conditions are used. For samespecies hybridization, moderately stringent to highly stringentconditions are used. The conditions can be empirically determined.

After successful amplification of the nucleic acid containing all or aportion of the identified sHASEGP polypeptide sequence or of a nucleicacid encoding all or a portion of a sHASEGP polypeptide homolog, thatsegment can be molecularly cloned and sequenced, and used as a probe toisolate a complete cDNA or genomic clone. This, in turn, permits thedetermination of the gene's complete nucleotide sequence, the analysisof its expression, and the production of its protein product forfunctional analysis. Once the nucleotide sequence is determined, an openreading frame encoding the sHASEGP polypeptide gene protein product canbe determined by any method well known in the art for determining openreading frames, for example, using publicly available computer programsfor nucleotide sequence analysis. Once an open reading frame is defined,it is routine to determine the amino acid sequence of the proteinencoded by the open reading frame. In this way, the nucleotide sequencesof the entire sHASEGP polypeptide genes as well as the amino acidsequences of sHASEGP polypeptide proteins and analogs can be identified.

Any eukaryotic cell potentially can serve as the nucleic acid source forthe molecular cloning of the sHASEGP polypeptide gene. The nucleic acidscan be isolated from vertebrate, mammalian, human, porcine, bovine,feline, avian, equine, canine, as well as additional primate sources,insects, plants and other organisms. The DNA can be obtained by standardprocedures known in the art from cloned DNA (e.g., a DNA “library”), bychemical synthesis, by cDNA cloning, or by the cloning of genomic DNA,or fragments thereof, purified from the desired cell (see, e.g.,Sambrook et al. 1989, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover,D. M. Ed., 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U. K. Vol. 1, 11. Clones derived from genomic DNA can containregulatory and intron DNA regions in addition to coding regions; clonesderived from cDNA will contain only exon sequences. For any source, thegene is cloned into a suitable vector for propagation thereof.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene.

The DNA can be cleaved at specific sites using various restrictionenzymes.

Alternatively, one can use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, for example, bysonication. The linear DNA fragments then can be separated according tosize by standard techniques, including but not limited to, agarose andpolyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene can be accomplished in a number ofways.

For example, a portion of the sHASEGP polypeptide (of any species) gene(e.g., a PCR amplification product obtained as described above or anoligonucleotide having a sequence of a portion of the known nucleotidesequence) or its specific RNA, or a fragment thereof be purified andlabeled, and the generated DNA fragments can be screened by nucleic acidhybridization to the labeled probe (Benton and Davis, Science 196: 180(1977); Grunstein and Hogness, Proc. Natl. Acad. Sci. U.S.A. 72: 3961(1975)). Those DNA fragments with substantial homology to the probe willhybridize. It is also possible to identify the appropriate fragment byrestriction enzyme digestion (s) and comparison of fragment sizes withthose expected according to a known restriction map if such is availableor by DNA sequence analysis and comparison to the known nucleotidesequence of sHASEGP polypeptide. Further selection can be carried out onthe basis of the properties of the gene. Alternatively, the presence ofthe gene can be detected by assays based on the physical, chemical, or-immunological properties of its expressed product. For example, cDNAclones, or DNA clones which hybrid-select the proper mRNA can beselected which produce a protein that, e.g., has similar or identicalelectrophoretic migration, isoelectric focusing behavior, proteolyticdigestion maps, antigenic properties, Hyaluronidase activity. If ananti-sHASEGP polypeptide antibody is available, the protein can beidentified by binding of labeled antibody to the putatively sHASEGPpolypeptide synthesizing clones, in an ELISA (enzyme-linkedimmunosorbent assay)-type procedure.

Alternatives to isolating the sHASEGP polypeptide genomic DNA include,but are not limited to, chemically synthesizing the gene sequence from aknown sequence or making cDNA to the mRNA that encodes the sHASEGPpolypeptide.

For example, RNA for cDNA cloning of the sHASEGP polypeptide gene can beisolated from cells expressing the protein. The identified and isolatednucleic acids then can be inserted into an appropriate cloning vector. Alarge number of vector-host systems known in the art can be used.Possible vectors include, but are not limited to, plasmids or modifiedviruses, but the vector system must be compatible with the host cellused. Such vectors include, but are not limited to, bacteriophages suchas lambda derivatives, or plasmids such as pBR322 or pUC plasmidderivatives or the Bluescript vector (Stratagene, La Jolla, Calif.). Theinsertion into a cloning vector can, for example, be accomplished byligating the DNA fragment into a cloning vector that has complementarycohesive termini.

If the complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules can beenzymatically modified. Alternatively, any site desired can be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers can include specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and sHASEGPpolypeptide gene can be modified by homopolymeric tailing.

Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, calciumprecipitation and other methods, so that many copies of the genesequence are generated.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated sHASEGP polypeptide gene,cDNA, or synthesized DNA sequence enables generation of multiple copiesof the gene.

Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

E. Vectors, Plasmids and Cells that Contain Nucleic Acids Encoding asHASEGP Polypeptide or Hyaluronidase Domain Thereof and Expression ofsHASEGP Polypeptides Vectors and Cells

For recombinant expression of one or more of the sHASEGP polypeptides,the nucleic acid containing all or a portion of the nucleotide sequenceencoding the sHASEGP polypeptide can be inserted into an appropriateexpression vector i.e., a vector that contains the necessary elementsfor the transcription and translation of the inserted protein codingsequence. The necessary transcriptional and translational signals canalso be supplied by the native promoter for sHASEGP genes, and/or theirflanking regions.

Also provided are vectors that contain nucleic acid encoding thesHASEGPs that can be introduced into an expression system capable ofproducing a soluble neutral active sHASEGP.

Cells containing the vectors are also provided. The cells includeeukaryotic and prokaryotic cells, and the vectors suitable for usetherein.

Eukaryotic cells, including dihydroflate reductase deficient ChineseHamster Ovary Cells (DG44), containing the vectors are provided.Suitable cells include yeast cells, fungal cells, plant cells, insectcells and animal cells. The cells are used to produce a sHASEGPpolypeptide or Hyaluronidase domain thereof by (a) growing theabove-described cells under conditions whereby the encoded sHASEGPpolypeptide or Hyaluronidase domain of the sHASEGP polypeptide isexpressed by the cell, and then (b) recovering the expressedHyaluronidase domain protein. In the exemplified embodiments, theHyaluronidase domain is secreted into the medium.

In one embodiment, the vectors include a sequence of nucleotides thatencodes a polypeptide that has Hyaluronidase activity and contains allor a portion of only the Hyaluronidase domain, or multiple copiesthereof, of a sHASEGP protein are provided. Also provided are vectorsthat comprise a sequence of nucleotides that encodes the Hyaluronidasedomain and additional portions of a sHASEGP protein up to and includinga full-length sHASEGP protein, as well as multiple copies thereof, arealso provided. The vectors can be selected for expression of the sHASEGPprotein or Hyaluronidase domain thereof in the cell or such that thesHASEGP protein is expressed as a secreted protein. Alternatively, thevectors can include signals necessary for secretion of encoded proteins.When the Hyaluronidase domain is expressed the nucleic acid is linked tonucleic acid encoding a secretion signal, such as the Saccharomycescerevisiae a mating factor signal sequence or a portion thereof, or thenative signal sequence.

In order to generate a soluble, neutral active sHASEGP, cells capable ofintroducing N-linked glycosylation are required. In the preferredembodiment, mammalian Chinese Hamster Ovary cells deficient indihydrofolate reductase such as DG44, are electroporated with a plasmidencoding a strong mammalian promoter, such as CMV, nucleic acid encodinga sHASEGP followed by an internal ribosomal entry site, the mousedihydrofolate reductase gene and the SV40 polyadenylation sequence asshown in SEQ ID NO 51. Such cells are then cultured in chemicallydefined medium in the absence of hypoxanthine and thymidine, followed byfurther gene amplification with increasing concentrations ofmethotrexate.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus e.g. vaccinia virus, adenovirus, etc.;insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.Note that bacterial expression of sHASEGP DNA will not result in acatalytically active sHASEGP per se, but when combined with properglycosylation machinery can be artificially glycosylated as such.

Any methods known to those of skill in the art for the insertion ofnucleic acid fragments into a vector can be used to construct expressionvectors containing a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding sHASEGP polypeptide, ordomains, derivatives, fragments or homologs thereof, can be regulated bya second nucleic acid sequence so that the genes or fragments thereofare expressed in a host transformed with the recombinant DNA molecule(s). For example, expression of the proteins can be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for sHASEGP polypeptide. Promoterswhich can be used include but are not limited to the SV40 early promoter(Bernoist and Chambon, Nature 290: 304-310 (1981) the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.,Cell 22: 787-797 (1980) the herpes thymidine kinase promoter (Wagner etal. Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981) the regulatorysequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the β-Lactamasepromoter (Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 1978)) Or The TAC Promoter Deboer et al., Proc. Natl. Acad.Sci. USA 80: 21-25 (1983)); see also “Useful Proteins from RecombinantBacteria”: in Scientific American 242: 79-94 (1980)); plant expressionvectors containing the opaline synthetase promoter (Herrar-Estrella etal., Nature 303: 209-213 (1984)) or the cauliflower mosaic virus 35S RNApromoter (Gardner et al., Nucleic Acids Res. 9: 2871 (1981)), and thepromoter of the photosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., Nature 310: 115-120 (1984)); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38: 639-646 (1984); Ornitzet al., Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986);Macdonald, Hepatology 7: 425-515 (1987)); insulin gene control regionwhich is active in pancreatic beta cells (Hanahan et al., Nature 315:115-122 (1985)), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., Cell 38: 647-658 (1984); Adams etal., Nature 318: 533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444 (1987)), mouse mammary tumor virus control region which isactive in testicular, breast, lymphoid and mast cells (Leder et al.,CELL 45: 485-495 (1986)), albumin gene control region which is active inliver (Pinkert et al., Genes and Devel. 1: 268-276 (1987)),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol. 5: 1639-1648 (1985); Hammer et al., Science235: 53-58 1987)), alpha-1 antitrypsin gene control region which isactive in liver (Kelsey et al., Genes And Devel. 1: 161-171 (1987)),beta globin gene control region which is active in myeloid cells (Mogramet al., Nature 315: 338-340 (1985); Kollias et al., CE//46: 89-94(1986)), myelin basic protein gene control region which is active inoligodendrocyte cells of the brain (Readhead et al., Cell 48: 703-712(1987)), myosin light chain-2 gene control region which is active inskeletal muscle (Sani, Nature 314: 283-286 (1985)), and gonadotrophicreleasing hormone gene control region which is active in gonadotrophs ofthe hypothalamus (Mason et al., Science 234: 1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a sHASEGP polypeptide, or adomain, fragment, derivative or homolog, thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e. G., anantibiotic resistance gene).

Specific initiation signals may also be required for efficienttranslation of a sHASEGP sequence. These signals include the ATGinitiation codon and adjacent sequences. In cases where sHASEGP, itsinitiation codon and upstream sequences are inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only coding sequence, ora portion thereof, is inserted, exogenous transcriptional controlsignals including the ATG initiation codon must be provided.Furthermore, the initiation codon must be in the correct reading frameto ensure transcription of the entire insert. Exogenous transcriptionalelements and initiation codons can be of various origins, both naturaland synthetic. The efficiency of expression may be enhanced by theinclusion of enhancers appropriate to the cell system in use (Scharf Det al (1994) Results Probl Cell Differ 20:125-62; Bittner et al (1987)Methods in Enzymol 153:516-544).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO (DG44, DXB11 CHO-K1), HeLa,MDCK, 293, WI38, etc have specific cellular machinery and characteristicmechanisms for such post-translational activities and may be chosen toensure the correct modification and processing of the introduced,foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably expresssHASEGP may be transformed using expression vectors which contain viralorigins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced sequences.Resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler M et al (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy I et al (1980) Cell 22:817-23) geneswhich can be employed in TK- or APRT-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, DHFR which confers resistance tomethotrexate (Wigler M et al (1980) Proc Natl Acad Sci 77:3567-70); npt,which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin F et al (1981) J Mol Biol 150:1-14) and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman S C and R C Mulligan(1988) Proc Natl Acad Sci 85:8047-51). Recently, the use of visiblemarkers has gained popularity with such markers as anthocyanins, betaglucuronidase and its substrate, GUS, and luciferase and its substrate,luciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes C A et al (1995)Methods Mol Biol 55:121-131).

Identification of Transformants Containing the Polynucleotide Sequence

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of anactive sHASEGP should be confirmed. For example, if the sHASEGP isinserted within a marker gene sequence, recombinant cells containingsHASEGP can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with a sHASEGPsequence under the control of a single promoter. Expression of themarker gene in response to induction or selection usually indicatesexpression of the tandem sHASEGP as well. Detection of a properlyglycosylated neutral active sHASEGP can be determined by way of testingthe conditioned media for sHASEGP enzyme activity under appropriateconditions.

Purification of sHASEGP

Host cells transformed with a sHASEGP nucleotide sequence may becultured under conditions suitable for the expression and recovery ofthe encoded protein from cell culture. The protein produced by arecombinant cell is preferably secreted but may be containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining sHASEGP can be designed with signal sequences that facilitatedirect secretion of sHASEGP through a prokaryotic or eukaryotic cellmembrane. Other recombinant constructions may join sHASEGP to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins (Kroll D J et al (1993) DNA Cell Biol12:441-53; cf discussion of vectors infra containing fusion proteins).

sHASEGP may also be expressed as a recombinant protein with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequences such as Factor XAor enterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and sHASEGP is useful to facilitate purification. One suchexpression vector provides for expression of a fusion proteincompromising a sHASEGP and contains nucleic acid encoding 6 histidineresidues followed by thioredoxin and an enterokinase cleavage site. Thehistidine residues facilitate purification on IMIAC (immobilized metalion affinity chromatography as described in Porath et al (1992) ProteinExpression and Purification 3: 263-281) while the enterokinase cleavagesite provides a means for purifying the chemokine from the fusionprotein.

In addition to recombinant production, fragments of sHASEGP may beproduced by direct peptide synthesis using solid-phase techniques (cfStewart et al (1969) Solid-Phase Peptide Synthesis, W H Freeman Co, SanFrancisco; Merrifield J (1963) J Am Chem Soc 85:2149-2154). In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster CityCalif.) in accordance with the instructions provided by themanufacturer. Various fragments of sHASEGP may be chemically synthesizedseparately and combined using chemical methods to produce thefull-length molecule.

Expression vectors containing the coding sequences, or portions thereof,of a sHASEGP polypeptide, is made, for example, by subcloning the codingportions into the EcoRl restriction site of each of the three PGEXvectors (glutathione S-transferase expression vectors (Smith andJohnson, Gene 7: 31-40 (1988)). This allows for the expression ofproducts in the correct reading frame. Exemplary vectors and systems forexpression of the Hyaluronidase domains of the sHASEGP polypeptidesinclude the well-known Pichia vectors (available, for example, fromInvitrogen, San Diego, Calif.), particularly those designed forsecretion of the encoded proteins. The protein can also be expressedcytoplasmically, such as in the inclusion bodies. One exemplary vectoris described in the examples.

Plasmids for transformation of E. coli cells, include, for example, thepET expression vectors (see, U.S. Pat. No. 4,952,496; available fromNovagen, Madison, Wis.; see, also literature published by Novagendescribing the system).

Such plasmids include pET 11a, which contains the T7lac promoter, T7terminator, the inducible E. coli lac operator, and the lac repressorgene; pET 12A-C, which contains the T7 promoter, T7 terminator, and theE. COLI OMPT secretion signal; and pET 15B and PET19B (Novagen, Madison,Wi), which contain a His-Tag leader sequence for use in purificationwith a His column and a thrombin cleavage site that permits cleavagefollowing purification over the column; the T7-lac promoter region andthe T7 terminator.

The vectors are introduced into host cells, such as Pichia cells andbacterial cells, such as E. coli, and the proteins expressed therein.Exemplary Pichia strains include, for example, GS115. Exemplarybacterial hosts contain chromosomal copies of DNA encoding T7 RNApolymerase operably linked to an inducible promoter, such as the LACUVpromoter (see, U.S. Pat. No. 4,952,496). Such hosts include, but are notlimited to, the lysogenic E. coli strain BL21 (DE3).

The sHASEGP domains, derivatives and analogs can be produced by variousmethods known in the art. For example, once a recombinant cellexpressing a sHASEGP polypeptide, or a domain, fragment or derivativethereof, is identified, the individual gene product can be isolated andanalyzed. This is achieved by assays based on the physical and/orfunctional properties of the protein, including, but not limited to,radioactive labeling of the product followed by analysis by gelelectrophoresis, immunoassay, cross-linking to marker-labeled product,and assays of proteolytic activity.

The sHASEGP polypeptides can be isolated and purified by standardmethods known in the art (either from natural sources or recombinanthost cells expressing the complexes or proteins), including but notrestricted to column chromatography (E.g., ion exchange, affinity, gelexclusion, reversed-phase high pressure and fast protein liquid),differential centrifugation, differential solubility, or by any otherstandard technique used for the purification of proteins.

In one embodiment, a sHASEGP can be purified to homogeneity from thechemically defined conditioned media of HZ24 transfected andmethotrexate amplified DG44 cells by 1) tangential flow diafiltration,2) binding and elution from anion exchange chromatography, 3) flowthrough phenyl sepharose chromatography, 4) binding and elution fromphenylboronate chromatography and 4) binding and elution withhydroxyapatite chromatography.

Functional properties can be evaluated using any suitable assay known inthe art.

Alternatively, once a sHASEGP polypeptide or its domain or derivative isidentified, the amino acid sequence of the protein can be deduced fromthe nucleotide sequence of the gene that encodes it. As a result, theprotein or its domain or derivative can be synthesized by standardchemical methods known in the art (e. G. see Hunkapiller et al., Nature310: 105-111 (1984)) followed by glycosylation in vitro.

Manipulations of sHASEGP polypeptide sequences can be made at theprotein level. Also contemplated herein are sHASEGP polypeptideproteins, domains thereof, derivatives or analogs or fragments thereof,which are differentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, pegylation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to an antibody molecule or other cellular ligand.

Any of numerous chemical modifications can be carried out by knowntechniques, including but not limited to specific chemical cleavage bycyanogen bromide, trypsin, chymotrypsin, papain, V8, NABH4, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin and other such agents.

In addition, domains, analogs and derivatives of a sHASEGP polypeptidecan be chemically synthesized. For example, a peptide corresponding to aportion of a sHASEGP polypeptide, which includes the desired domain orwhich mediates the desired activity in vitro can be synthesized by useof a peptide synthesizer.

Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the sHASEGPpolypeptide sequence. Non-classical amino acids include but are notlimited to the D-isomers of the common amino acids, a-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, E-ABU, e-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionoicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, ca-methyl amino acids, na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be d (dextrorotary) or l (levorotary).

In cases where natural products are suspected of being mutant or areisolated from new species, the amino acid sequence of the sHASEGPpolypeptide isolated from the natural source, as well as those expressedin vitro, or from synthesized expression vectors in vivo or in vitro,can be determined from analysis of the DNA sequence, or alternatively,by direct sequencing of the isolated protein. Such analysis can beperformed by manual sequencing or through use of an automated amino acidsequenator.

Modifications—A variety of modifications of the sHASEGP polypeptides anddomains are contemplated herein. A sHASEGP-encoding nucleic acidmolecule can be modified by any of numerous strategies known in the art(Sambrook ET A/. (1990), Molecular Cloning, A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). The sequencescan be cleaved at appropriate sites with restriction endonuclease (s),followed by further enzymatic modification if desired, isolated, andligated in vitro. In the production of the gene encoding a domain,derivative or analog of sHASEGP, care should be taken to ensure that themodified gene retains the original translational reading frame,uninterrupted by translational stop signals, in the gene region wherethe desired activity is encoded.

Additionally, the sHASEGP-encoding nucleic acid molecules can be mutatedin vitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy pre-existingones, to facilitate further in vitro modification. Also, as describedherein muteins with primary sequence alterations, such as replacementsof Cys residues and elimination or addition of glycosylation sites arecontemplated; the sHASEGP of SEQ ID No. 1 has seven potentialglycosylation sites. Such mutations can be effected by any technique formutagenesis known in the art, including, but not limited to, chemicalmutagenesis and in vitro site-directed mutagenesis (Hutchinson et al.,j. Biol. Chem. 253: 6551-6558 (1978)), use of TABE Linkers (Pharmacia).In one embodiment, for example, a sHASEGP polypeptide or domain thereofis modified to include a fluorescent label. In other specificembodiments, the sHASEGP polypeptide is modified to have aheterobifunctional reagent, such heterobifunctional reagents can be usedto crosslink the members of the complex.

In addition, domains, analogs and derivatives of a sHASEGP can bechemically synthesized. For example, a peptide corresponding to aportion of a sHASEGP, which includes the desired domain or whichmediates the desired activity in vitro can be synthesized by use of apeptide synthesizer. Furthermore, if desired, nonclassical amino acidsor chemical amino acid analogs can be introduced as a substitution oraddition into the sHASEGP sequence. Non-classical amino acids includebut are not limited to the D-isomers of the common amino acids, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, S-ABU,e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionoic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as ti-methyl amino acids, ca-methyl aminoacids, na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be d (dextrorotary) or l (levorotary).

F. Generation of a Functionally Active Glycosylated sHASEGP withN-Linked Sugar Moieties

Properly N-glycosylated human sHASEGP is required to generate acatalytically stable protein. N-linked glycosylation of sHASEGP can beachieved through various techniques. Glycosylation of sHASEGP can beachieved by introducing nucleic acids encoding sHASEGP into cells ofeukaryotic origin capable of proper N-linked glycosylation oralternatively, by contacting sHASEGP polypeptide with cell free extractsor purified enzymes capable of introducing the desired N-linked sugarmoieties.

Selection of an Expression System

Eukaryotic cell expression systems vary in the extent and type ofglycosylation they introduce into an ectopically expressed polypeptide.CHO cells are, for example, highly efficient at the introduction ofN-linked glycosylation into an active sHASEGP polypeptide.

Additional eukaryotic expression systems that introduce N-linkedglycosylation to generate a functional sHASEGP product can be tested byintroducing a human sHASEGP expression plasmid into said cells andtesting for neutral activity. Proper N-linked glycosylation can bedetermined by way of FACE analysis of PNGASE released oligosaccharides.Glycosylation profiles of catalytically active sHASEGP's are furtherprovided herein. Verification of glycosylation can also be made bytreatment of sHASEGP from said cells with PNGASE-F or by growth of suchcells in tunicamycin following introduction of sHASEGP encoding nucleicacids.

N-glycosylation of sHASEGP polypeptide in vitro. The sHASEGP polypeptidecan be N-glycosylated by contact of sHASEGP polypeptide with cell-freeextracts containing activity capable of transferring N-linked sugars tosHASEGP polypeptide such as canine microsomal membranes or throughcoupled transcription and translation as is commercially available(Promega Madison Wis.).

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right. All oligosaccharides describedherein are described with the name or abbreviation for the non-reducingsaccharide (e.g., Gal), followed by the configuration of the glycosidicbond (.alpha. or. beta.), the ring bond, the ring position of thereducing saccharide involved in the bond, and then the name orabbreviation of the reducing saccharide (e.g., GlcNAc). The linkagebetween two sugars may be expressed, for example, as 2,3, 2.fw darw.3,or (2,3). Each saccharide is a pyranose.

As used herein, N-linked sugar moiety refers to an oligosaccharideattached to a sHASEGP via the amide nitrogen of Asn residues. N-linkedoligosaccharides fall into several major types (oligomannose, complex,hybrid, sulfated), all of which have (Man) 3-GlcNAc-GlcNAc-coresattached via the amide nitrogen of Asn residues that fall within-Asn-Xaa-Thr/Ser-sequences (where Xaa is not Pro). N-linked sites areoften indirectly assigned by the appearance of a “blank” cycle duringsequencing. Positive identification can be made after release of theoligosaccharide by PNGase F, which converts the glycosylated Asn to Asp.After PNGase F release, N-linked oligosaccharides can be purified usingBio-Gel P-6 chromatography, with the oligosaccharide pool subjected topreparative high pH anion exchange chromatography (HPAEC) (Townsend etal., (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide isomerscan be resolved using HPAEC. Fucose residues will shift elutionpositions earlier in the HPAEC chromatogram, while additional sialicacid residues will increase the retention time. Concurrent treatment ofglycoproteins whose oligosaccharide structures are known (e.g., bovinefetuin, a-l acid glycoprotein, ovalbumin, RNAse B, transferrin) canfacilitate assignment of the oligosaccharide peaks. The collectedoligosaccharides can be characterized by a combination of compositionaland methylation linkage analyses (Waegheet al., (1983) Carbohydr Res.123, 281-304.), with anomeric configurations assigned by NMRspectroscopy (Van Halbeek (1993) in Methods Enzymol 230).

Alternatively, oligosaccharides can be identified by fluorescenceassisted carbohydrate electrophoresis (FACE) Callewaert et al. (2001)Glycobiology 11, 275-281.

G. Detection and Characterization of N-Linked Sugar Moieties on sHASEGP

Determining whether a protein is in fact glycosylated is the initialstep in glycoprotein glycan analysis. Polyacrylamide gel electrophoresisin the presence of sodium dodecyl sulfate (SDS-PAGE) has become themethod of choice as the final step prior to protein sequencing.Glycosylated proteins often migrate as diffuse bands by SDS-PAGE. Amarked decrease in bandwidth and change in migration position aftertreatment with peptide-N4-(N-acetyl-D-glucosaminyl) asparagine amidase(PNGase F) is considered diagnostic of N-linked glycosylation. If theother types of glycosylation are predominant other approaches must beused. Lectin blotting methods provide an approach that is independent ofthe class of glycosylation (N versus O). Lectins, carbohydrate-bindingproteins from various plant tissues, have both high affinity and narrowspecificity for a wide range of defined sugar epitopes found onglycoprotein glycans (Cummings, R. D. (1994) Methods in Enzymol. 230,66-86.). When conjugated with biotin or digoxigenin, they can be easilyidentified on membrane blots through a colorimetric reaction utilizingavidin or anti-digoxigenin antibodies conjugated with alkalinephosphatase (Haselbeck et al., (1993) Methods in Mol. Biol. 14,161-173.), analogous to secondary antibody-alkaline phosphatasereactions employed in Western blotting. Screening with a panel oflectins with well-defined specificity can provide considerableinformation about a glycoprotein's carbohydrate complement. Importantly,the color development amplification is sufficiently high that 10-50 ngof a glycoprotein can easily be seen on a membrane blot of an SDS-PAGE.Although lectins exhibit very high affinity for their cognate ligands,some do reveal significant avidity for structurally related epitopes.Thus, it is important to carefully note the possibility ofcross-reactivity when choosing a panel of lectins, and apply those withthe highest probability of individually distinguishing complex, hybridand high mannose N-linked glycans from O-linked structures.

Monosaccharide analysis can also be used to determine whether sHASEGP isglycosylated and as in the case of lectin analysis provides additionalinformation on structural features. Quantitative monosaccharidecomposition analysis i) identifies glycosylated proteins, ii) gives themolar ratio of individual sugars to protein, iii) suggests, in somecases, the presence of oligosaccharide classes, iv) is the first step indesigning a structural elucidation strategy, and v) provides a measureof production consistency for recombinant glycoprotein therapeutics. Inrecent years high-pH anion-exchange chromatography with pulsedamperometric detection (HPAEC-PAD) has been extensively used todetermine monosaccharide composition (Townsend et al., (1995) inCarbohydrate Analysis: High-performance liquid chromatography andcapillary electrophoresis (Z. El Rassi ed.). pp. 181-209.). Morerecently, fluorophore-based labeling methods have been introduced andmany are available in kit form. A distinct advantage of fluorescentmethods is an increase in sensitivity (50-fold). One potentialdisadvantage is that different monosaccharides may demonstrate differentselectivity for the fluorophore during the coupling reaction, either inthe hydrolysate or in the external standard mixture. However, theincrease in sensitivity and the ability to identify whichmonosaccharides are present from a small portion of the total amount ofavailable glycoprotein, as well as the potential for greater sensitivityusing laser induced fluorescence makes this approach attractive.

Monosaccharide composition analysis of small amounts of sHASEGP is bestperformed on PVDF (PSQ) membranes, after either electroblotting(Weitzhandleret al., (1993) J. Biol. Chem. 268, 5121-5130.) or ifsmaller aliquots are to be analyzed on dot blots. PVDF is an idealmatrix for carbohydrate analysis since neither mono- or oligosaccharidesbind to the membrane, once released by either acid or enzymatichydrolysis.

FACE analysis is an efficient means of detecting glycosylation profilesof sHASEGP's. FACE® N-Linked Oligosaccharide Profiling (Prozyme) with30% oligosaccharide gels is one such mechanism. Oligosaccharides cleavedfrom 100 μg of glycoproteins by enzymatic digestion with N-Glycanase(a.k.a PNGase), labeled using the fluorophore ANTS, and separated byelectrophoresis can be used for detection of sHASEGP glycosylationprofiles. The relative positions of the oligosaccharide bands aredetermined by running the sample and dilutions of the sample alongsidean oligosaccharide standard ladder which designated the migrationdistance in Degree of Polymerization (DP) units.

H. Screening Methods to Identify Compounds that Modulate sHASEGPActivity

Several types of assays are exemplified and described herein. It isunderstood that the Hyaluronidase domains can be used in other assays.It is shown here, however, that the Hyaluronidase domains exhibitcatalytic activity.

As such they are ideal for in vitro screening assays.

They can also be used in binding assays.

The sHASEGP full length zymogens, activated enzymes, and Hyaluronidasedomains are contemplated for use in any screening assay known to thoseof skill in the art, including those provided herein. Hence thefollowing description, if directed to Hyaluronidase assays is intendedto apply to use of a single chain Hyaluronidase domain or acatalytically active portion thereof of any Hyaluronidase, including asHASEGP. Other assays, such as binding assays are provided herein,particularly for use with a sHASEGP, including any variants, such assplice variants thereof.

1. Catalytic Assays for identification of agents that modulate theHyaluronidase activity of a sHASEGP protein. Methods for identifying amodulator of the catalytic activity of a sHASEGP, particularly a singlechain Hyaluronidase domain or catalytically active portion thereof, areprovided herein. The methods can be practiced by: contacting thesHASEGP, a full-length zymogen or activated form, and particularly asingle-chain domain thereof, with a substrate of the sHASEGP in thepresence of a test substance, and detecting the proteolysis of thesubstrate, whereby the activity of the sHASEGP is assessed, andcomparing the activity to a control. For example, a control can be theactivity of the sHASEGP assessed by contacting a sHASEGP, including afull-length zymogen or activated form, and particularly a single-chaindomain thereof, particularly a single-chain domain thereof, with asubstrate of the sHASEGP, and detecting the proteolysis of thesubstrate, whereby the activity of the sHASEGP is assessed. The resultsin the presence and absence of the test compounds are compared. Adifference in the activity indicates that the test substance modulatesthe activity of the sHASEGP. Activators of sHASEGP activation cleavageare also contemplated; such assays are discussed below.

In one embodiment a plurality of the test substances are screenedsimultaneously in the above screening method. In another embodiment, thesHASEGP is isolated from a target cell as a means for then identifyingagents that are potentially specific for the target cell.

In another embodiment, a test substance is a therapeutic compound, andwhereby a difference of the sHASEGP activity measured in the presenceand in the absence of the test substance indicates that the target cellresponds to the therapeutic compound.

One method includes the steps of (a) contacting the sHASEGP polypeptideor Hyaluronidase domain thereof with one or a plurality of testcompounds under conditions conducive to interaction between the ligandand the compounds; and (b) identifying one or more compounds in theplurality that specifically binds to the ligand.

Another method provided herein includes the steps of a) contacting asHASEGP polypeptide or Hyaluronidase domain thereof with a substrate ofthe sHASEGP polypeptide, and detecting the degradation of substrate,whereby the activity of the sHASEGP polypeptide is assessed; b)contacting the sHASEGP polypeptide with a substrate of the sHASEGPpolypeptide in the presence of a test substance, and detecting thedegradation of the substrate, whereby the activity of the sHASEGPpolypeptide is assessed; and c) comparing the activity of the sHASEGPpolypeptide assessed in steps a) and b), whereby the activity measuredin step a) differs from the activity measured in step b) indicates thatthe test substance modulates the activity of the sHASEGP polypeptide.

In another embodiment, a plurality of the test substances is screenedsimultaneously. In comparing the activity of a sHASEGP polypeptide inthe presence and absence of a test substance to assess whether the testsubstance is a modulator of the sHASEGP polypeptide, it is unnecessaryto assay the activity in parallel, although such parallel measurement istypical. It is possible to measure the activity of the sHASEGPpolypeptide at one time point and compare the measured activity to ahistorical value of the activity of the sHASEGP polypeptide.

For instance, one can measure the activity of the sHASEGP polypeptide inthe presence of a test substance and compare with historical value ofthe activity of the sHASEGP polypeptide measured previously in theabsence of the test substance, and vice versa. This can be accomplished,for example, by providing the activity of the sHASEGP polypeptide on aninsert or pamphlet provided with a kit for conducting the assay.

Methods for selecting substrates for a particular sHASEGP are describedin the EXAMPLES, and particular Hyaluronidase assays are exemplified.

Combinations and kits containing the combinations optionally includinginstructions for performing the assays are provided. The combinationsinclude a sHASEGP polypeptide and a substrate of the sHASEGP polypeptideto be assayed; and, optionally reagents for detecting proteolysis of thesubstrate. The substrates, which can be chromogenic or fluorogenicmolecules, including glycosaminoglycans, subject to proteolysis by aparticular sHASEGP polypeptide, can be identified empirically by testingthe ability of the sHASEGP polypeptide to cleave the test substrate.Substrates that are cleaved most effectively i.e. at the lowestconcentrations and/or fastest rate or under desirable conditions), areidentified.

Additionally provided herein is a kit containing the above-describedcombination. The kit optionally includes instructions for identifying amodulator of the activity of a sHASEGP polypeptide. Any sHASEGPpolypeptide is contemplated as target for identifying modulators of theactivity thereof.

2. Binding assays. Also provided herein are methods for identificationand isolation of agents, particularly compounds that bind to sHASEGPs.The assays are designed to identify agents that bind to the isolatedHyaluronidase domain (or a protein, other than a sHASEGP polypeptide,that contains the Hyaluronidase domain of a sHASEGP polypeptide), and tothe activated form, including the activated form derived from thefull-length zymogen or from an extended Hyaluronidase domain. Theidentified compounds are candidates or leads for identification ofcompounds for treatments of disorders and diseases involving aberrantHyaluronidase activity. The sHASEGP polypeptides used in the methodsinclude any sHASEGP polypeptide as defined herein, including the sHASEGPsingle chain Hyaluronidase domain or proteolytically active portionthereof.

A variety of methods are provided herein. These methods can be performedin solution or in solid phase reactions in which the sHASEGP polypeptide(s) or Hyaluronidase domain (s) thereof are linked, either directly orindirectly via a linker, to a solid support. Screening assays aredescribed in the Examples, and these assays have been used to identifycandidate compounds.

For purposes herein, all binding assays described above are provided forsHASEGP.

Methods for identifying an agent, such as a compound, that specificallybinds to a sHASEGP single chain Hyaluronidase domain, a full-lengthactivated sHASEGP or two chain Hyaluronidase domain thereof are providedherein. The method can be practiced by (a) contacting the sHASEGP withone or a plurality of test agents under conditions conducive to bindingbetween the sHASEGP and an agent; and (b) identifying one or more agentswithin the plurality that specifically binds to the sHASEGP.

For example, in practicing such methods the sHASEGP polypeptide is mixedwith a potential binding partner or an extract or fraction of a cellunder conditions that allow the association of potential bindingpartners with the polypeptide. After mixing, peptides, polypeptides,proteins or other molecules that have become associated with a sHASEGPare separated from the mixture. The binding partner that bound to thesHASEGP can then be removed and further analyzed. To identify andisolate a binding partner, the entire protein, for instance the entiredisclosed protein of SEQ ID No. 1 can be used. Alternatively, a fragmentof the protein can be used.

A variety of methods can be used to obtain cell extracts or body fluids,such as blood, serum, urine, sweat, synovial fluid, CSF and other suchfluids.

For example, cells can be disrupted using either physical or chemicaldisruption methods. Examples of physical disruption methods include, butare not limited to, sonication and mechanical shearing. Examples ofchemical lysis methods include, but are not limited to, detergent lysisand enzyme lysis. A skilled artisan can readily adapt methods forpreparing cellular extracts in order to obtain extracts for use in thepresent methods.

Once an extract of a cell is prepared, the extract is mixed with thesHASEGP under conditions in which association of the protein with thebinding partner can occur. A variety of conditions can be used,including conditions that resemble conditions found in the cytoplasm ofa human cell or in a body fluid, such as blood. Features, such asosmolarity pH, temperature, and the concentration of cellular extractused, can be varied to optimize the association of the protein with thebinding partner. Similarly, methods for isolation of molecules ofinterest from body fluids are known.

After mixing under appropriate conditions, the bound complex isseparated from the mixture. A variety of techniques can be used toseparate the mixture. For example, antibodies specific to a sHASEGP canbe used to immunoprecipitate the binding partner complex. Alternatively,standard chemical separation techniques such as chromatography anddensity/sediment centrifugation can be used.

After removing the non-associated cellular constituents in the extract,the binding partner can be dissociated from the complex usingconventional methods. For example, dissociation can be accomplished byaltering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixedextract, the sHASEGP can be immobilized on a solid support. For example,the protein can be attached to a nitrocellulose matrix or acrylic beads.Attachment of the protein or a fragment thereof to a solid support aidsin separating peptide/binding partner pairs from other constituentsfound in the extract. The identified binding partners can be either asingle protein or a complex made up of two or more proteins.

Alternatively, the nucleic acid molecules encoding the single chainHyaluronidases can be used in a yeast two-hybrid system. The yeasttwo-hybrid system has been used to identify other protein partner pairsand can readily be adapted to employ the nucleic acid molecules hereindescribed.

Another in vitro binding assay, particularly for a sHASEGP, uses amixture of a polypeptide that contains at least the catalytic domain ofone of these proteins and one or more candidate binding targets orsubstrates. After incubating the mixture under appropriate conditions,the ability of the sHASEGP or a polypeptide fragment thereof containingthe catalytic domain to bind to or interact with the candidate substrateis assessed. For cell-free binding assays, one of the componentsincludes or is coupled to a detectable label. The label can provide fordirect detection, such as radioactivity, luminescence, optical orelectron density, etc., or indirect detection such as an epitope tag, anenzyme, etc. A variety of methods can be employed to detect the labeldepending on the nature of the label and other assay components. Forexample, the label can be detected bound to the solid substrate or aportion of the bound complex containing the label can be separated fromthe solid substrate, and the label thereafter detected.

3. Detection of signal transduction sHASEGP, which is a membraneanchored protein, can be involved directly or indirectly in signaltransduction directly as a cell surface receptor or indirectly byactivating proteins, such as pro-growth factors that can initiate signaltransduction.

In addition, secreted sHASEGP, such as the soluble domain of sHASEGP asdescribed in SEQ ID NO. 4, can be involved in signal transduction eitherdirectly by binding to or interacting with a cell surface receptor orindirectly by activating proteins, such as pro-growth factors that caninitiate signal transduction. Assays for assessing signal transductionare well known to those of skill in the art, and can be adapted for usewith the sHASEGP polypeptide.

Assays for identifying agents that affect or alter signal transductionmediated directly or indirectly, such as via activation of a pro-growthfactor, by a sHASEGP, particularly the full length or a sufficientportion to anchor the extracellular domain or a functional portionthereof of a sHASEGP on the surface of a cell are provided. Such assays,include, for example, transcription based assays in which modulation ofa transduced signal is assessed by detecting an effect on an expressionfrom a reporter gene (see, e.g., U.S. Pat. No. 5,436,128).

4. Methods for Identifying Agents that Modulate the Expression a NucleicAcid encoding a sHASEGP. Another embodiment provides methods foridentifying agents that modulate the expression of a nucleic acidencoding a sHASEGP. Such assays use any available means of monitoringfor changes in the expression level of the nucleic acids encoding asHASEGP.

Assay formats can be used to monitor the ability of the agent tomodulate the expression of a nucleic acid encoding a sHASEGP. Forinstance, mRNA expression can be monitored directly by hybridization tothe nucleic acids. Also enzyme assays as described can be used to detectagents that modulate the expression of sHASEGP.

Cell lines are exposed to the agent to be tested under appropriateconditions and time and total RNA or mRNA is isolated by standardprocedures (see, e.g. Sambrook et al (1989) MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed. Cold Spring Harbor Laboratory Press). Probesto detect differences in RNA expression levels between cells exposed tothe agent and control cells can be prepared from the nucleic acids. Itis typical, but not necessary, to design probes which hybridize onlywith target nucleic acids under conditions of high stringency. Onlyhighly complementary nucleic acid hybrids form under conditions of highstringency. Accordingly, the stringency of the assay conditionsdetermines the amount of complementarity that should exist between twonucleic acid strands in order to form a hybrid. Stringency should bechosen to maximize the difference in stability between the probe: targethybrid and potential probe: non-target hybrids.

For example, N- and C-terminal fragments of the sHASEGP can be expressedin bacteria and used to search for proteins that bind to thesefragments. Fusion proteins, such as His-tag or GST fusion to the N- orC-terminal regions of the sHASEGP can be prepared for use as asubstrate. These fusion proteins can be coupled to, for example,Glutathione-Sepharose beads and then probed with cell lysates or bodyfluids. Prior to lysis, the cells or body fluids can be treated with acandidate agent that can modulate a sHASEGP or proteins that interactwith domains thereon. Lysate proteins binding to the fusion proteins canbe resolved by SDS-PAGE, isolated and identified by protein sequencingor mass spectroscopy, as is known in the art.

Antibody probes are prepared by immunizing suitable mammalian hosts inappropriate immunization protocols using the peptides, polypeptides orproteins if they are of sufficient length (e.g., 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more consecutive amino acidsthe sHASEGP polypeptide or if required to enhance immunogenicity,conjugated to suitable carriers. Methods for preparing immunogenicconjugates with carriers, such as bovine serum albumin (BSA), keyholelimpet hemocyanin (KLH), or other carrier proteins are well known in theart. In some circumstances, direct conjugation using, for example,carbodiimide reagents can be effective; in other instances linkingreagents such as those supplied by Pierce Chemical Co., Rockford, Ill.,can be desirable to provide accessibility to the hapten. Hapten peptidescan be extended at either the amino or carboxy terminus with a Cysresidue or interspersed with cysteine residues, for example, tofacilitate linking to a carrier.

Administration of the immunogens is conducted generally by injectionover a suitable time period and with use of suitable adjuvants, as isgenerally understood in the art. During the immunization schedule,titers of antibodies are taken to determine adequacy of antibodyformation.

Anti-peptide antibodies can be generated using synthetic peptidescorresponding to, for example, the carboxy terminal amino acids of thesHASEGP.

Synthetic peptides can be as small as 1-3 amino acids in length,generally at least 4 or more amino acid residues long. The peptides canbe coupled to KLH using standard methods and can be immunized intoanimals, such as rabbits or ungulates. Polyclonal antibodies can then bepurified, for example using Actigel beads containing the covalentlybound peptide.

While the polyclonal antisera produced in this way can be satisfactoryfor some applications, for pharmaceutical compositions, use ofmonoclonal preparations are generally used. Immortalized cell lineswhich secrete the desired monoclonal antibodies can be prepared usingthe standard method of Kohler et al., (Nature 256: 495-7 (1975)) ormodifications which effect immortalization of lymphocytes or spleencells, as is generally known. The immortalized cell lines secreting thedesired antibodies are screened by immunoassay in which the antigen isthe peptide hapten, polypeptide or protein.

When the appropriate immortalized cell culture secreting the desiredantibody is identified, the cells can be cultured either in vitro or byproduction in vivo via ascites fluid. Of particular interest, aremonoclonal antibodies that recognize the catalytic domain or activationcleavage site (region) of a sHASEGP.

The antibodies or fragments can also be produced. Regions that bindspecifically to the desired regions of receptor also can be produced inthe context of chimeras with multiple species origin.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed.

The agents can be, as examples, peptides, small molecules, andcarbohydrates. A skilled artisan can readily recognize that there is nolimit as to the structural nature of the agents.

The peptide agents can be prepared using standard solid phase (orsolution phase) peptide synthesis methods, as is known in the art. Inaddition, the DNA encoding these peptides can be synthesized usingcommercially available oligonucleotide synthesis instrumentation andproduced recombinantly using standard recombinant production systems.The production using solid phase peptide synthesis is necessitated ifnon-gene-encoded amino acids are to be included.

I. Methods of Treatment

sHASEGP's identified by the methods herein are used for treating orpreventing abnormal accumulations of sHASEGP substrates in an animal,particularly a mammal, including a human. In one embodiment, the methodincludes administering to a mammal an effective amount of a sHASEGPglycoprotein, whereby the disease or disorder is treated or prevented.

In another embodiment, a sHASEGP inhibitor can be used in the treatmentof an excess amount of neutral hyaluronidase activity. The mammaltreated can be a human. The inhibitors provided herein are thoseidentified by the screening assays. In addition, antibodies andantisense nucleic acids or double-stranded RNA (dsRNA), such as RNAi,are contemplated.

1. Antisense treatment: In a specific embodiment, as describedhereinabove, sHASEGP polypeptide function is reduced or inhibited bysHASEGP polypeptide antisense nucleic acids, to treat or preventexcessive chondroitinase activity. The therapeutic or prophylactic useof nucleic acids of at least six nucleotides, generally up to about 150nucleotides, that are antisense to a gene or cDNA encoding sHASEGPpolypeptide or a portion thereof is provided. A sHASEGP polypeptide“antisense” nucleic acid as used herein refers to a nucleic acid capableof hybridizing to a portion of a sHASEGP polypeptide RNA (generallymRNA) by virtue of some sequence complementarity, and generally underhigh stringency conditions. The antisense nucleic acid can becomplementary to a coding and/or noncoding region of a sHASEGPpolypeptide mRNA. Such antisense nucleic acids have utility astherapeutics that reduce or inhibit sHASEGP polypeptide function, andcan be used in the treatment or prevention of disorders as describedsupra.

The sHASEGP polypeptide antisense nucleic acids are of at least sixnucleotides and are generally oligonucleotides (ranging from 6 to about150 nucleotides including 6 to 50 nucleotides). The antisense moleculecan be complementary to all or a portion of the Hyaluronidase domain.For example, the oligonucleotide is at least 10 nucleotides, at least 15nucleotides, at least 100 nucleotides, or at least 125 nucleotides. Theoligonucleotides can be DNA or RNA or chimeric mixtures or derivativesor modified versions thereof, single-stranded or double-stranded. Theoligonucleotide can be modified at the base moiety, sugar moiety, orphosphate backbone. The oligonucleotide can include other appendinggroups such as peptides, or agents facilitating transport across thecell membrane (see e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA86: 6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648-652 (1987); PCT Publication No. WO 88/09810, published Dec. 15,1988) or blood-brain barrier (see e.g., PCT Publication No. WO 89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents (seee.g., Krol et al., BioTechniques 6: 958-976 (1988)) or intercalatingagents (see e.g., Zon. Pharm. Res. 5: 539-549 (1988)).

The sHASEGP polypeptide antisense nucleic acid generally is anoligonucleotide, typically single-stranded DNA or RNA or an analogthereof or mixtures thereof. For example, the oligonucleotide includes asequence antisense to a portion of a nucleic acid that encodes a humansHASEGP polypeptide. The oligonucleotide can be modified at any positionon its structure with substituents generally known in the art.

The sHASEGP polypeptide antisense oligonucleotide can include at leastone modified base moiety which is selected from the group including, butnot limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-apos-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-n-2-carboxypropyl)uracil, (ACP3) w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide includes at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose. Theoligonucleotide can include at least one modified phosphate backboneselected from a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

The oligonucleotide can be an a-anomeric oligonucleotide. An a-anomericoligonucleotide forms specific double-stranded hybrids withcomplementary RNA in which the strands run parallel to each other(Gautier et al., Nucl. Acids Res. 15: 6625-6641 (1987)).

The oligonucleotide can be conjugated to another molecule, such as, butare not limited to, a peptide; hybridization triggered cross-linkingagent, transport agent or a hybridization-triggered cleavage agent. Theoligonucleotides can be synthesized by standard methods known in theart, e.g. by use of an automated DNA synthesizer (such as arecommercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al., Nucl. Acids Res. 16: 3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. USA85: 7448-7451 (1988)), etc. In a specific embodiment, the sHASEGPpolypeptide antisense oligonucleotide includes catalytic RNA or aribozyme (see, e.g., PCT International Publication WO 90/11364,published Oct. 4, 1990; Sarver et al., Science 247: 1222-1225 (1990)).In another embodiment, the oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15: 6131-6148(1987)), or a chimeric RNA-DNA analogue Inoue et al., FEBS Lett. 215:327-330 (1987)).

Alternatively, the oligonucleotide can be double-stranded RNA (dsRNA)such as RNAi.

In an alternative embodiment, the sHASEGP polypeptide antisense nucleicacid is produced intracellularly by transcription from an exogenoussequence.

For example, a vector can be introduced in vivo such that it is taken upby a cell, within which cell the vector or a portion thereof istranscribed, producing an antisense nucleic acid (RNA). Such a vectorwould contain a sequence encoding the sHASEGP polypeptide antisensenucleic acid. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others known in the art, used for replication and expression inmammalian cells. Expression of the sequence encoding the sHASEGPpolypeptide antisense RNA can be by any promoter known in the art to actin mammalian, including human, cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, Nature 290: 304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell 22: 787-797 (1980), the herpes thymidinekinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature 296: 39-42 (1982), etc.

The antisense nucleic acids include sequence complementary to at least aportion of an RNA transcript of a sHASEGP polypeptide gene, including ahuman sHASEGP polypeptide gene. Absolute complementarity is notrequired. The amount of sHASEGP polypeptide antisense nucleic acid thatis effective in the treatment or prevention of neoplastic diseasedepends on the nature of the disease, and can be determined empiricallyby standard clinical techniques.

Where possible, it is desirable to determine the antisense cytotoxicityin cells in vitro, and then in useful animal model systems prior totesting and use in humans.

2. RNA interference RNA interference (RNAi) (see, e.g. Chuang et al.(2000) Proc. Natl. Acad. Sci. USA 97: 4985) can be employed to inhibitthe expression of a gene encoding a sHASEGP. Interfering RNA (RNAi)fragments, particularly double-stranded (ds) RNAi, can be used togenerate loss-of-sHASEGP function. Methods relating to the use of RNAito silence genes in organisms including, mammals, C. elegans, Drosophilaand plants, and humans are known (see, e.g., Fire et al. (1998) Nature391: 806-811; Fire (1999) Trends Genet. 15: 358-363; Sharp (2001) GenesDev. 15: 485-490; Hammond et al. (2001) Nature Rev, Genet. 2: 110-119;Tuschl (2001) Chem. Biochem. 2: 239-245; Hamilton et al. (1999) Science286: 950-952; Hammond et al. (2000) Nature 404: 293-296; Zamore et al.(2000) Cell 101: 25-33; Bernstein et al. (2001) Nature 409: 363-366;Elbashir et al. (2001) Genes Dev. 15: 188-200; Elbashir et al. (2001)Nature 411: 494-498; International PCT application No. WO 01/29058;International PCT application No. WO 99/32619).

Double-stranded RNA (dsRNA)-expressing constructs are introduced into ahost, such as an animal or plant using, a replicable vector that remainsepisomal or integrates into the genome. By selecting appropriatesequences, expression of dsRNA can interfere with accumulation ofendogenous mRNA encoding a sHASEGP. RNAi also can be used to inhibitexpression in vitro.

Regions include at least about 21 (or 21) nucleotides that are selective(i.e. unique) for sHASEGP are used to prepare the RNAi. Smallerfragments of about 21 nucleotides can be transformed directly (i.e., invitro or in vivo) into cells; larger RNAi dsRNA molecules are generallyintroduced using vectors that encode them. dsRNA molecules are at leastabout 21 bp long or longer, such as 50, 100, 150, 200 and longer.Methods, reagents and protocols for introducing nucleic acid moleculesinto cells in vitro and in vivo are known to those of skill in the art.

3. Gene Therapy in an exemplary embodiment, nucleic acids that include asequence of nucleotides encoding a sHASEGP polypeptide or functionaldomains or derivative thereof, are administered to promote sHASEGPpolypeptide function, by way of gene therapy. Gene therapy refers totherapy performed by the administration of a nucleic acid to a subject.In this embodiment, the nucleic acid produces its encoded protein thatmediates a therapeutic effect by promoting sHASEGP polypeptide function.Any of the methods for gene therapy available in the art can be used(see, Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu,Biotherapy 3: 87-95 (1991); Tolstoshev, An. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan andAnderson, An. Rev. Biochem. 62: 191-217 (1993); TIBTECH 11 5: 155-215(1993). For example, one therapeutic composition for gene therapyincludes a sHASEGP polypeptide-encoding nucleic acid that is part of anexpression vector that expresses a sHASEGP polypeptide or domain,fragment or chimeric protein thereof in a suitable host. In particular,such a nucleic acid has a promoter operably linked to the sHASEGPpolypeptide coding region, the promoter being inducible or constitutive,and, optionally, tissue-specific. In another particular embodiment, anucleic acid molecule is used in which the sHASEGP polypeptide codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the sHASEGP protein nucleicacid (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 8932-8935(1989); Zijlstra et al., Nature 342: 435-438 (1989)).

Delivery of the nucleic acid into a patient can be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, J. Biol.Chem, 262: 4429-4432 (1987)) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand is afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. In yet another embodiment, the nucleicacid can be targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (see, e.g., PCT Publications WO 92/06180dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilsonet al.); WO 92/20316 dated Nov. 26, 1992 (Findeis et al.); WO 93/14188dated Jul. 22, 1993 (Clarke et al.), WO 93/20221 dated Oct. 14, 1993(Young)). Alternatively, the nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci.USA 86: 8932-8935 (1989); Zijistra et al., Nature 342: 435-438 (1989)).

In a specific embodiment, a viral vector that contains the sHASEGPpolypeptide nucleic acid is used. For example, a retroviral vector canbe used (see Miller et al., Meth. Enzymol. 217: 581-599 (1993)). Theseretroviral vectors have been modified to delete retroviral sequencesthat are not necessary for packaging of the viral genome and integrationinto host cell DNA. The sHASEGP polypeptide nucleic acid to be used ingene therapy is cloned into the vector, which facilitates delivery ofthe gene into a patient. More detail about retroviral vectors can befound in Boesen et al., Biotherapy 6: 291-302 (1994), which describesthe use of a retroviral vector to deliver the mdr1 gene to hematopoieticstem cells in order to make the stem cells more resistant tochemotherapy.

Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., J. Clin. Invest. 93: 644-651 (1994); Kiem etal., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human GeneTherapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. InGenetics And Devel. 3: 110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3: 499-503 (1993) present a reviewof adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genesto the respiratory epithelia of rhesus monkeys. Other instances of theuse of adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252: 431-434 (1991); Rosenfeld et al., Cell 68: 143-155 (1992);and Mastrangeli et al., J. Clin. Invest. 91: 225-234 (1993).

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204: 289-300 (1993).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, Meth. Enzymol.217: 599-618 (1993); Cohen et al., Meth. Enzymol. 217: 618-644 (1993);Cline, Pharmac. Ther. 29: 69-92 (1985)) and can be used, provided thatthe necessary developmental and physiological functions of the recipientcells are not disrupted. The technique should provide for the stabletransfer of the nucleic acid to the cell, so that the nucleic acid isexpressible by the cell and generally heritable and expressible by itscell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In an embodiment, epithelial cells areinjected, e.g., subcutaneously. In another embodiment, recombinant skincells can be applied as a skin graft onto the patient. Recombinant bloodcells (e.g., hematopoietic stem or progenitor cells) can be administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., suchas stem cells obtained from bone marrow, umbilical cord blood,peripheral blood, fetal liver, and other sources thereof.

For example, a cell used for gene therapy is autologous to the patient.In an embodiment in which recombinant cells are used in gene therapy, asHASEGP polypeptide nucleic acid is introduced into the cells such thatit is expressible by the cells or their progeny, and the recombinantcells are then administered in vivo for therapeutic effect. In aspecific embodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells that can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment.

Such stem cells include but are not limited to hematopoietic stem cells(HSC), stem cells of epithelial tissues such as the skin and the liningof the gut, embryonic heart muscle cells, liver stem cells (PCTPublication WO 94/08598, dated Apr. 28, 1994), and neural stem cells(Stemple and Anderson, Cell 71: 973-985 (1992)).

Epithelial stem cells (ESC) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, Meth. Cell Bio. 21A: 229 (1980)). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESC or keratinocytes obtained from the skin or lining of the gutof a patient or donor can be grown in tissue culture (Rheinwald, Meth.Cell Bio. 21A: 229 (1980); Pittelkow and Scott, Cano. Clinic Proc. 61:771 (1986)). If the ESC are provided by a donor, a method forsuppression of host versus graft reactivity (e.g., irradiation, drug orantibody administration to promote moderate immunosuppression) also canbe used.

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment. Techniques by which this can beaccomplished include (a) the isolation and establishment of HSC culturesfrom bone marrow cells isolated from the future host, or a donor, or (b)the use of previously established long-term HSC cultures, which can beallergenic or xenogenic.

Non-autologous HSC generally are used with a method of suppressingtransplantation immune reactions of the future host/patient. In aparticular embodiment, human bone marrow cells can be obtained from theposterior iliac crest by needle aspiration (see, e.g., Kodo et al., J.Clin. Invest. 73: 1377-1384 (1984)). For example, the HSC can be madehighly enriched or in substantially pure form. This enrichment can beaccomplished before, during, or after long-term culturing, and can bedone by any techniques known in the art. Long-term cultures of bonemarrow cells can be established and maintained by using, for example,modified Dexter cell culture techniques (Dexter et al., J. Cell Physiol.91: 335 (1977)) or Witlock-Witte culture techniques (Witlock and Witte,Proc. Natl. Acad. Sci. USA 79: 3608-3612 (1982)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy includes an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

3. Prodrugs—A method for treating tumors is provided. The method ispracticed by administering a prodrug that is cleaved at a specific siteby a HASEGP to release an active drug or precursor that can be convertedto active drug in vivo. Upon contact with a cell that expresses sHASEGPactivity, the prodrug is converted into an active drug. The prodrug canbe a conjugate that contains the active agent, such as an anti-tumordrug, such as a cytotoxic agent, or other therapeutic agent (TA), linkedto a substrate for the targeted sHASEGP, such that the drug or agent isinactive or unable to enter a cell, in the conjugate, but is activatedupon cleavage. The prodrug, for example, can contain an chondroitinsulfate molecule, typically a relatively short, less than about 20disaccharide units, that is catalytically cleaved by the targetedsHASEGP. Cytotoxic agents, include, but are not limited to, alkylatingagents, antiproliferative agents and tubulin binding agents. Othersinclude, vinca drugs, mitomycins, bleomycins and taxanes.

J. Pharmaceutical Compositions and Modes of Administration

1. Components of the Compositions.

Pharmaceutical compositions containing an active sHASEGP are providedherein. Also provided are combinations of compounds that modulate theactivity of a sHASEGP polypeptide and another treatment or compound fortreatment of a hyaluronidase disorder, such as an antibody compound.

The sHASEGP polypeptide and a second agent can be packaged as separatecompositions for administration together or sequentially orintermittently. Alternatively, they can be provided as a singlecomposition for administration or as two compositions for administrationas a single composition. The combinations can be packaged as kits.

2. Formulations and Route of Administration

The sHASEGP polypeptides and soluble human hyaluronidase domain thereofprovided herein can be formulated as pharmaceutical compositions,typically for single dosage administration. The concentrations of thepolypeptides in the formulations are effective for delivery of anamount, upon administration, that is effective for the intendedtreatment. Typically, the composition's are formulated for single dosageadministration. To formulate a composition, the weight fraction of asHASEGP polypeptide, soluble human hyaluronidase domains thereof ormixture thereof is dissolved, suspended, dispersed or otherwise mixed ina selected vehicle at an effective concentration such that the treatedcondition is relieved or ameliorated.

Pharmaceutical carriers or vehicles suitable for administration of thesHASEGP or soluble human hyaluronidase domains thereof provided hereininclude any such carriers known to those skilled in the art to besuitable for the particular mode of administration.

In addition, the polypeptides can be formulated as the solepharmaceutically active ingredient in the composition or can be combinedwith other active ingredients. Liposomal suspensions, includingtissue-targeted liposomes, can also be suitable as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art. For example, liposome formulations can beprepared as described in U.S. Pat. No. 4,522,811.

The active sHASEGP or soluble human hyaluronidase domain thereof isincluded in the pharmaceutically acceptable carrier in an amountsufficient to exert a therapeutically useful effect in the absence ofundesirable side effects on the patient treated. The therapeuticallyeffective concentration can be determined empirically by testing thepolypeptides in known in vitro and in vivo systems such as by using theassays provided herein or see, e.g., Taliani et al. (1996) Anal.Biochem. 240: 60-67, Filocamo et al. (1997) J. Virology 71: 1417-1427,Sudo et al. (1996) Antiviral Res. 32: 9-18, Buffard et al. (1995)Virology 209: 52-59, Bianchi et al. (1996) Anal. Biochem. 237: 239-244,Hamatake et al. (1996) Intervirology 39:249-258, Steinkühler et al.(1998) Biochem. 37:8899-8905, D'Souza et al. (1995) J. Gen. Virol.76:1729-1736, Takeshita et al. (1997) Anal. Biochem. 247: 242-246; seealso, e.g., Shimizu et al. (1994) J. Virol. 68: 8406-8408; Mizutani etal. (1996) J. Virol. 70: 7219-7223, Mizutani et al. (1996) Biochem.Biophys. Res. Commun. 227: 822-826, Lu et al. (1996) Proc. Natl. Acad.Sci. (USA) 93: 1412-1417, Hahm et al. (1996) Virology 226: 318-326, Itoet al. (1996) J. Gen. Virol. 77: 1043-1054, Mizutani et al. (1995)Biochem. Biophys. Res. Commun. 212: 906-911, Cho et al. (1997) J. Virol.Meth. 65: 201-207 and then extrapolated therefrom for dosages forhumans.

Typically a therapeutically effective dosage is contemplated. Theamounts administered can be on the order of 0.001 to 1 mg/ml, includingabout 0.005-0.05 mg/ml and about 0.01 mg/ml, of blood volume.Pharmaceutical dosage unit forms are prepared to provide from about 1 mgto about 1000 mg, including from about 10 to about 500 mg, and includingabout 25-75 mg of the essential active ingredient or a combination ofessential ingredients per dosage unit form. The precise dosage can beempirically determined.

In some instances, a high Unit dose of human sHASEGP is preferable. Forexample, with intravenous administration of sHASEGP concentrations ofsHASEGP from 500-100,000 Units per ml are preferable. Lyophilizedformulations of sHASEGP are also ideal for storage of large Unit dosesof sHASEGP. 200,000 Unit lyophilized vials of sHASEGP are contemplatedfor intravenous delivery.

High concentration doses are also contemplated for the delivery of smallvolumes of sHASEGP. Administration of 10-100 ul of 5000 Units/ml sHASEGPis contemplated for injection in the anterior chamber to dissolve preadministered viscoelastic substances during cataract and phakicintraocular lens implantation surgeries. Small volume injections of50-200 U/ml doses are also contemplated for intravitreal procedures suchas the treatment of vitreous hemorrhage or vitro-retinal detachment indiabetic retinopathy.

The active ingredient can be administered at once, or can be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and can be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuescan also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or use of the claimed compositions andcombinations containing them.

Pharmaceutically acceptable derivatives include acids, salts, esters,hydrates, solvates and prodrug forms. The derivative is typicallyselected such that its pharmacokinetic properties are superior to thecorresponding neutral sHASEGP or soluble human hyaluronidase domainthereof.

Thus, effective concentrations or amounts of one or more of thepolypeptides herein or pharmaceutically acceptable derivatives thereofare mixed with a suitable pharmaceutical carrier or vehicle forsystemic, topical or local administration to form pharmaceuticalcompositions. sHASEGP polypeptides or soluble human hyaluronidasedomains thereof are included in an amount effective for ameliorating ortreating the disorder for which treatment is contemplated. Theconcentration of active polypeptide in the composition depends onabsorption, inactivation, excretion rates of the active polypeptide, thedosage schedule, amount administered, particular formulation as well asother factors known to those of skill in the art.

The therapeutic agents for use in the methods can be administered by anyroute known to those of skill in the art, such as, but are not limitedto, topically, intraarticularly, intracisternally, intraocularly,intraventricularly, intrathecally, intravenously, intramuscularly,intraperitoneally, intradermally, intratracheally, as well as by anycombination of any two or more thereof. Dry powder pulmonaryformulations can be envisioned as well.

The most suitable route for administration will vary depending upon theproposed use, such as, for example, use as a delivery agent tofacilitate subcutaneous delivery of fluids, use to reduce intraocularpressure in the eyes of glaucoma patients receiving viscoelastics or useas a “spreading agent” to enhance the activity of chemotherapeutics, andthe location of interest, such as a particular internal organ, a tumorgrowth, intraocular cavity and the epidermis. Modes of administrationinclude, but are not limited to, topically, locally, intraarticularly,intracisternally, intraocularly, intraventricularly, intrathecally,intravenously, intramuscularly, intratracheally, intraperitoneally,intradermally, and by a combination of any two or more thereof. Forexample, for treatment of various cancers, such as squamous cellcarcinoma, breast cancer, urinary bladder cancer and gastrointestinalcancer, local administration, including administration to the site ofthe tumor growth (e.g., intrathecally, intraventricularly, orintracisternally) provides the advantage that the therapeutic agent canbe administered in a high concentration without risk of thecomplications that can accompany systemic administration of atherapeutic agent.

Pharmaceutical and cosmetic carriers or vehicles suitable foradministration of the sHASEGP polypeptides or soluble humanhyaluronidase domain thereof provided herein include any such carriersknown to those skilled in the art to be suitable for the particular modeof administration. In addition, the polypeptides can be formulated asthe sole pharmaceutically active ingredient in the composition or can becombined with other active ingredients that do not impair the desiredaction, or with materials that supplement the desired action known tothose of skill in the art. For example, the sHASEGP polypeptidesprovided herein can be used as a delivery or “spreading” agent incombination with a second active compound, such as a therapeuticallyeffective agent, including, but not limited to a drug or a prodrug, tofacilitate delivery of or to enhance the activity of the second activeingredient. In a particular embodiment, a sHASEGP polypeptide or asoluble human hyaluronidase domain thereof can be co-formulated with ananesthetic agent, such as Lignocaine, Bupivicaine or a mixture of thetwo, and, optionally, a hormonal agent, such as epinephrine, to decreaseor stop blood uptake during ophthalmic surgery. A sHASEGP polypeptide ora soluble human hyaluronidase domain thereof can also be co-formulatedwith various chemotherapeutics, such as a toxin and a tumor necrosisfactor, to enhance the activity of the chemotherapeutic and/or theaccessibility of the target tumors to the chemotherapeutic. The activecompound is included in the carrier in an amount sufficient to exert atherapeutically useful effect in the absence of serious toxic effects onthe treated individual. The effective concentration can be determinedempirically by testing the compounds using in vitro and in vivo systems,including the animal models described herein.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent, such as water for injection, saline solution, fixedoil, polyethylene glycol, glycerine, propylene glycol or other syntheticsolvent; antimicrobial agents, such as benzyl alcohol and methylparabens; antioxidants, such as ascorbic acid and sodium bisulfite;cheating agents, such as ethylenediaminetetraacetic acid (EDTA);buffers, such as acetates, citrates and phosphates; and agents for theadjustment of tonicity, including, but not limited to sodium chloride,calcium chloride, magnesium chloride, dextrose, glycerol or boric acid.Parenteral preparations can be enclosed in ampoules, disposable syringesor single or multiple dose vials made of glass, plastic or othersuitable material.

The sHASEGP polypeptides or soluble human hyaluronidase domains thereofcan be suspended in micronized or other suitable form or can bederivatized to produce a more soluble active product or to produce aprodrug. The form of the resulting mixture depends upon a number offactors, including the intended mode of administration and thesolubility of the polypeptide in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the targetedcondition and can be empirically determined using methods known to thoseof skill in the art. To formulate a composition, the weight fraction ofpolypeptide is dissolved, suspended, dispersed, or otherwise mixed in aselected vehicle at an effective concentration such that the targetedcondition is relieved or ameliorated.

In instances in which the sHASEGP polypeptides or soluble humanhyaluronidase domain thereof exhibit insufficient solubility, methodsfor solubilizing polypeptides can be used. Such methods are known tothose of skill in this art, and include, but are not limited to, usingcosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such asTWEEN® and Pluronic® F68; or dissolution in aqueous sodium bicarbonate.Derivatives of the polypeptides, such as prodrugs of the polypeptidescan also be used in formulating effective pharmaceutical compositions.For ophthalmic indications, the compositions are formulated in anophthalmically acceptable carrier. For the ophthalmic uses herein, localadministration, either by topical administration or by injection iscontemplated. Time-release formulations are also desirable. Typically,the compositions are formulated for single dosage administration, sothat a single dose administers an effective amount.

Upon mixing or addition of the polypeptide with the vehicle, theresulting mixture can be a solution, suspension, emulsion or othercomposition and can be formulated as an aqueous mixtures, a creams,gels, ointments, emulsions, solutions, elixirs, lotions, suspensions,tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories,bandages, or any other formulation suitable for systemic, topical orlocal administration.

The form of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of thecompound in the selected carrier or vehicle. If necessary,pharmaceutically acceptable salts or other derivatives of the compoundsare prepared. For local internal administration, such as, intramuscular,parenteral or intra-articular administration, the compounds arepreferably formulated as a solution suspension in an aqueous-basedmedium, such as isotonically buffered saline or are combined with abiocompatible support or bioadhesive intended for internaladministration.

The sHASEGP polypeptide or soluble human hyaluronidase domain thereof isincluded in the pharmaceutically acceptable carrier in an amountsufficient to exert a therapeutically useful effect in the absence ofundesirable side effects on the patient treated. It is understood thatnumber and degree of side effects depends upon the condition for whichthe compounds are administered. For example, certain toxic andundesirable side effects are tolerated when treating life-threateningillnesses that would not be tolerated when treating disorders of lesserconsequence. Amounts effective for therapeutic use will, of course,depend on the severity of the disease and the weight and general stateof the subject as well as the route of administration. Localadministration of the therapeutic agent will typically require a smallerdosage than any mode of systemic administration, although the localconcentration of the therapeutic agent can, in some cases, be higherfollowing local administration than can be achieved with safety uponsystemic administration.

Since individual subjects can present a wide variation in severity ofsymptoms and each therapeutic agent has its unique therapeuticcharacteristics, it is up to the practitioner to determine the responseof a subject to treatment and vary the dosages accordingly. Dosages usedin vitro can provide useful guidance in the amounts useful for in situadministration of the pharmaceutical composition, and animal models canin some cases be used to determine effective dosages for treatment ofparticular disorders. In general, however, for local administration, itis contemplated that an effective amount of the therapeutic agent willbe an amount within the range from about 0.1 picograms (pg) up to about1 ng per kg body weight. Various considerations in arriving at aneffective amount are known to those of skill in the art and aredescribed (see, e.g., Goodman And Gilman's: The Pharmacological Bases ofTherapeutics, 8th ed., Pergamon Press, 1990; Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990; and Mantyhet al., (Science, 278: 275-79, 1997) involving the intrathecal injectionof a neuronal specific ligand-toxin, each of which is hereinincorporated by reference in its entirety).

The formulations of the sHASEGP polypeptides or soluble humanhyaluronidase domains thereof for use herein include those suitable fororal, rectal, topical, inhalational, buccal (e.g., sublingual),parenteral (e.g., subcutaneous, intramuscular, intradermal, orintravenous), transdermal administration or any route. The most suitableroute in any given case depends on the nature and severity of thecondition being treated and on the nature of the particular activecompound that is being used. The formulations are provided foradministration to humans and animals in unit dosage forms, such astablets, capsules, pills, powders, granules, sterile parenteralsolutions or suspensions, and oral solutions or suspensions, andoil-water emulsions containing suitable quantities of the polypeptidesand/or other agents or pharmaceutically acceptable derivatives thereof.The pharmaceutical therapeutically active polypeptides and/or otheragents and derivatives thereof are typically formulated and administeredin unit-dosage forms or multiple-dosage forms. A unit-dose form as usedherein refers to physically discrete units suitable for human and animalsubjects and packaged individually as is known in the art.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the sHASEGP polypeptide or soluble humanhyaluronidase domain thereof and, optionally, another agent orpharmaceutically acceptable derivatives thereof. The pharmaceuticallytherapeutically active compounds and derivatives thereof are typicallyformulated and administered in unit-dosage forms or multiple-dosageforms. A unit-dose form as used herein refers to physically discreteunits suitable for human and animal subjects and packaged individuallyas is known in the art. Each unit-dose contains a predetermined quantityof the therapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms include, butare not limited to, ampoules, syringes and individually packaged tabletsor capsules. For example, a small volume formulation containing astabilized solution with 1 to 5000 Units of sHASEGP in a small volume,such as 5 to 50 μl, can be prepackaged in a syringe for use, such asafter viscoelastic injection. Unit-dose forms can be administered infractions or multiples thereof. A multiple-dose form is a plurality ofidentical unit-dosage forms packaged in a single container to beadministered in segregated unit-dose form. Examples of multiple-doseforms include vials, bottles of tablets or capsules or bottles of pintsor gallons. Hence, multiple dose form is a multiple of unit-doses thatare not segregated in packaging.

The composition can contain along with the active ingredient, such as asHASEGP polypeptide: a diluent, such as lactose, sucrose, dicalciumphosphate, or carboxymethylcellulose; a lubricant, such as magnesiumstearate, calcium stearate and talc; and a binder such as starch,natural gums, such as gum acaciagelatin, glucose, molasses,polyvinylpyrrolidine, celluloses and derivatives thereof, povidone,crospovidones and other such binders known to those of skill in the art.Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered can also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, or solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents. Methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art (see e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition, 1975). The composition or formulation to be administeredcontains a quantity of the active compound in an amount sufficient toalleviate the symptoms of the treated subject. For example, a standardstabilized formulation of sHASEGP or a soluble human hyaluronidasedomain thereof as provided herein includes 150 Units/ml of the solubleglycoprotein formulated in EDTA, NaCl and CaCl₂. Additionally, ananti-bacterial or anti-fungal agent, including, but not limited tothiomersal, can be present in the formulation. Another formulationprovided herein is a stabilized solution or lyophilized form of sHASEGPor a soluble human hyaluronidase domain thereof in EDTA, NaCl and CaCl₂,containing an effective active amount of the soluble glycoprotein, suchas 150 Unit/ml, with the addition of lactose, such as 13 mg/ml. Alsoprovided herein is a formulation containing a stabilized solution orlyophilized form of sHASEGP or a soluble human hyaluronidase domainthereof in EDTA, NaCl and CaCl₂ containing an effective active amount ofthe soluble glycoprotein, such as 150 Unit/ml, with the addition oflactose, such as 13 mg/ml, and Albumin, Pluronic® F68, TWEEN® and/orother detergent. Another formulation provided herein, either lyophilizedor as a stabilized solution, contains an effect amount of sHASEGP or asoluble human hyaluronidase domain thereof, such as 1 to 300 Units/ml,in EDTA, NaCl and CaCl₂.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non-toxic carrier can beprepared. For oral administration, the pharmaceutical compositions cantake the form of, for example, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets can be coated by methods well known in theart.

The sHASEGPs or a soluble human hyaluronidase domain thereof orpharmaceutically acceptable derivatives can be prepared with carriersthat protect the soluble glycoprotein against rapid elimination from thebody, such as time release formulations or coatings. The compositionscan include other pharmaceutically effective agents known in the generalart to be of value in treating one or more of the diseases or medicalconditions, including, but not limited to, a chemotherapeutic agent, ananalgesic agent, an anti-inflammatory agent, an antimicrobial agent, anamoebicidal agent, a trichomonocidal agent, an anti-parkinson agent, ananti-malarial agent, an anticonvulsant agent, an anti-depressant agent,and antiarthritics agent, an anti-fungal agent, an antihypertensiveagent, antipyretic agent, an anti-parasite agent, an antihistamineagent, an alpha-adrenargic agonist agent, an alpha blocker agent, ananesthetic agent, a bronchi dilator agent, a biocide agent, abactericide agent, a bacteriostat agent, a betadrenergic blocker agent,a calcium channel blocker agent, a cardiovascular drug agent, acontraceptive agent, a decongestant agent, a diuretic agent, adepressant agent, a diagnostic agent, a electrolyte agent, a hypnoticagent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent,a muscle contractant agent, an ophthalmic agent, a parasympathomimeticagent, a psychic energizer agent, ophthalmic agent, aparasympathomimetic agent, a psychic energizer agent, a sedative agent,a sympathomimetic agent, a tranquilizer agent, an urinary agent, avaginal agent, a viricide agent, a vitamin agent, a non-steroidalanti-inflammatory agent, an angiotensin converting enzyme inhibitoragent, a polypeptide, a protein, a nucleic acid, a drug, a prodrug, aorganic molecule and a sleep inducer, to obtain desired combinations ofproperties. It is to be understood that such combination therapyconstitutes a further aspect of the compositions and methods oftreatment provided herein.

1. Compositions for Oral Administration

Oral pharmaceutical dosage forms are solid, gel or liquid. The soliddosage forms are tablets, capsules, granules, and bulk powders. Types oforal tablets include compressed, chewable lozenges and tablets, whichcan be enteric-coated, sugarcoated or film-coated. Capsules can be hardor soft gelatin capsules, while granules and powders can be provided innon-effervescent or effervescent form with the combination of otheringredients known to those skilled in the art.

The pharmaceutical compositions containing a sHASEGP or a soluble humanhyaluronidase domain thereof can be in liquid form, for example,solutions, syrups or suspensions, or can be presented as a drug productfor reconstitution with water or other suitable vehicle before use. Suchliquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid).

In certain embodiments, the formulations are solid dosage forms,preferably capsules or tablets. The tablets, pills, capsules, trochesand the like can contain any of the following ingredients, or compoundsof a similar nature: a binder; a diluent; a disintegrating agent; alubricant; a glidant; a sweetening agent; and a flavoring agent.

Examples of binders include microcrystalline cellulose, gum tragacanth,glucose solution, acacia mucilage, gelatin solution, sucrose and starchpaste. Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin, and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

If oral administration is desired, the sHASEGP or a soluble humanhyaluronidase domain thereof could be provided in a composition thatprotects it from the acidic environment of the stomach. For example, thecomposition can be formulated in an enteric coating that maintains itsintegrity in the stomach and releases the active compound in theintestine. The composition can also be formulated in combination with anantacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup can contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

The sHASEGP or a soluble human hyaluronidase domain thereof can also bemixed with other active materials which do not impair the desiredaction, or with materials that supplement the desired action, such asantacids, H2 blockers, and diuretics. The active ingredient is acompound or pharmaceutically acceptable derivative thereof as describedherein. Higher concentrations, up to about 98% by weight of the activeingredient can be included.

Pharmaceutically acceptable carriers included in tablets are binders,lubricants, diluents, disintegrating agents, coloring agents, flavoringagents, and wetting agents. Enteric-coated tablets, because of theenteric-coating, resist the action of stomach acid and dissolve ordisintegrate in the neutral or alkaline intestines. Sugar-coated tabletsare compressed tablets to which different layers of pharmaceuticallyacceptable substances are applied. Film-coated tablets are compressedtablets which have been coated with a polymer or other suitable coating.Multiple compressed tablets are compressed tablets made by more than onecompression cycle utilizing the pharmaceutically acceptable substancespreviously mentioned. Coloring agents can also be used in the abovedosage forms. Flavoring and sweetening agents are used in compressedtablets, sugar-coated, multiple compressed and chewable tablets.Flavoring and sweetening agents are especially useful in the formationof chewable tablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and can contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicadd, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Diluents include lactose and sucrose. Sweetening agentsinclude sucrose, syrups, glycerin and artificial sweetening agents suchas saccharin. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelauryl ether. Organic additives include citric and tartaric acid.Sources of carbon dioxide include sodium bicarbonate and sodiumcarbonate. Coloring agents include any of the approved certified watersoluble FD and C dyes, and mixtures thereof. Flavoring agents includenatural flavors extracted from plants such fruits, and synthetic blendsof compounds that produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is encapsulated ina gelatin capsule. Such solutions, and the preparation and encapsulationthereof, are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and4,410,545. For a liquid dosage form, the solution, e.g., for example, ina polyethylene glycol, can be diluted with a sufficient quantity of apharmaceutically acceptable liquid carrier, e.g., water, to be easilymeasured for administration.

Alternatively, liquid or semi-solid oral formulations can be prepared bydissolving or dispersing the sHASEGP or a soluble human hyaluronidasedomain thereof in vegetable oils, glycols, triglycerides, propyleneglycol esters (e.g., propylene carbonate) and other such carriers, andencapsulating these solutions or suspensions in hard or soft gelatincapsule shells. Other useful formulations include those set forth inU.S. Pat. Nos. Re 28,819 and 4,358,603.

Formulations suitable for buccal (sublingual) administration include,for example, lozenges containing the sHASEGP or a soluble humanhyaluronidase domain thereof in a flavored base, usually sucrose andacacia or tragacanth; and pastilles containing the compound in an inertbase such as gelatin and glycerin or sucrose and acacia.

In all embodiments, tablets and capsules formulations can be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they can becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

2. Injectables, Solutions and Emulsions

Parenteral administration of the sHASEGP or a soluble humanhyaluronidase domain thereof, generally characterized by injection,either subcutaneously, intramuscularly or intravenously is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions; solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.Suitable excipients are, for example, water, saline, dextrose, glycerolor ethanol. In addition, if desired, the pharmaceutical compositions tobe administered can also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,stabilizers, solubility enhancers, and other such agents, such as, forexample, sodium acetate, sorbitan monolaurate, triethanolamine oleateand cyclodextrins. Implantation of a slow-release or sustained-releasesystem, such that a constant level of dosage is maintained (see, e.g.,U.S. Pat. No. 3,710,795) is also contemplated herein. The percentage ofthe sHASEGP or a soluble human hyaluronidase domain thereof contained insuch parenteral compositions is dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent or sterile solution just prior to use, includinghypodermic tablets, sterile suspensions ready for injection, sterile dryinsoluble products ready to be combined with a vehicle just prior to useand sterile emulsions. The solutions can be either aqueous ornonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thiomersal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcelluose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN® 80). A sequestering or chelatingagent of metal ions includes EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration.Typically a therapeutically effective dosage is formulated to contain aconcentration of at least about 0.1% w/w up to about 90% w/w or more,preferably more than 1% w/w of the active compound to the treatedtissue(s). The active ingredient, such as a sHASEGP or a soluble humanhyaluronidase domain thereof, can be administered at once, or can bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the tissue being treated and can bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values can also vary with the age of theindividual treated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of theformulations, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed formulations.

The compounds provided herein can be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection can be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions can be suspensions, solutions oremulsions in oily or aqueous vehicles, and can contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the active ingredient can be in powder form forreconstitution with a suitable vehicle, e.g., sterile pyrogen-free wateror other solvents, before use. For example, provided herein areparenteral formulations containing an effective amount of sHASEGP or asoluble human hyaluronidase domain thereof, such as 500 to 500,000Units, in a stabilized solution or a lyophilized from.

The compound can be suspended in micronized or other suitable form orcan be derivatized to produce a more soluble active product or toproduce a prodrug. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and can be empirically determined.

3. Lyophilized Powders

Also provided herein are lyophilized powders containing sHASEGP or asoluble human hyaluronidase domain thereof, which can be reconstitutedfor administration as solutions, emulsions and other mixtures. Theseformulations can also be reconstituted and formulated as solids or gels.

The sterile, lyophilized powder is prepared by dissolving a solidportion of or mixing an aliquot of a solution containing a sHASEGP or asoluble human hyaluronidase domain thereof in a suitable solvent. Thesolvent can contain an excipient that improves the stability or otherpharmacological component of the powder or reconstituted solution,prepared from the powder. Excipients that can be used include, but arenot limited to, dextrose, sorbitol, fructose, corn syrup, xylitol,glycerin, glucose, sucrose, lactose or other suitable agent. The solventcan also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at,typically, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the lyophilized formulation.Generally, the solution resulting from the sterile filtration isapportioned into vials for lyophilization. Each vial can contain asingle dosage, such as 10-1000 mg or 100-500 mg, or multiple dosages ofthe compound.

Briefly, the lyophilized powder is prepared by dissolving dextrose,sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose,lactose or other suitable agent, about 1-20%, in a suitable buffer, suchas citrate, sodium or potassium phosphate or other such buffer known tothose of skill in the art at about neutral pH. Then, a selected salt,such as, for example, the sodium salt of the sHASEGP (about 1 gm of thesalt per 10-100 gms of the buffer solution, typically about 1 gm/30gms), is added to the resulting mixture above room temperature, such asat about 30-35° C., and stirred until it dissolves. The resultingmixture is diluted by adding more buffer, to decrease the resultingconcentration of the salt by about 10-50%, typically about 15-25%). Theresulting mixture is sterile filtered or treated to remove particulatesand to insure sterility, and apportioned into vials for lyophilization.The lyophilized powder can be stored under appropriate conditions, suchas at about 4 C to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, a therapeutically effective amount of the lyophilizedpowder containing a sHASEGP or a soluble human hyaluronidase domainthereof is added per milliliter of sterile water or other suitablecarrier. The precise amount depends upon the selected compound and canbe empirically determined by methods known to those of skill in the art.

4. Topical Administration

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture can be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The compositions of sHASEGP or a soluble human hyaluronidase domainthereof or pharmaceutically acceptable derivatives thereof can beformulated as aerosols for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatmentinflammatory diseases, particularly asthma). These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will typically have diameters ofless than 50 microns, such as less than 10 microns.

For administration by inhalation, the compositions for use herein can bedelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, including,but not limited to, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide and other suitable gases. Inthe case of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compositions can be formulated for local or topical application,such as for topical application to the skin and mucous membranes, suchas in the eye, in the form of gels, creams, and lotions and forapplication to the eye or for intracisternal or intraspinal application.Topical administration is contemplated for transdermal delivery and alsofor administration to the eyes or mucosa, or for inhalation therapies.Nasal solutions of the active compound alone or in combination withother pharmaceutically acceptable excipients can also be administered.

For example, formulations suitable for topical application to the skinor to the eye generally are formulated as an ointment, cream, lotion,paste, gel, spray, aerosol and oil. Carriers that can be used includevaseline, lanoline, polyethylene glycols, alcohols, and combinations oftwo or more thereof. The topical formulations can further advantageouslycontain 0.05 to 15 percent by weight of thickeners, including, but notlimited to, hydroxypropylmethylcellulose, methylcellulose,polyvinylpyrrolidone, polyvinyl alcohol, poly (alkylene glycols),poly/hydroxyalkyl, (meth)acrylates or poly (meth) acrylamides. A topicalformulation is often applied by instillation or as an ointment into theconjunctival sac. It also can be used for irrigation or lubrication ofthe eye, facial sinuses, and external auditory meatus. The topicalformulations in the liquid state can be also present in a hydrophilicthree-dimensional polymer matrix in the form of a strip, contact lens,and the like from which the active components are released. It can alsobe injected into the anterior eye chamber and other places. For example,provided herein is a formulation for intraocular use after viscoelasticinjecting containing a stabilized solution of an effective amount of asHASEGP or a soluble human hyaluronidase domain thereof, such as 1 to5000 Units of the soluble glycoprotein with 30 to 150,000 Units/mg ofspecific activity, in a small volume, such as 5 to 50 μl.

These solutions, particularly those intended for ophthalmic use, can beformulated as 0.01%-10% isotonic solutions, pH about 5-7, withappropriate salts.

5. Compositions for Other Routes of Administration

Other routes of administration, such as topical application, transdermalpatches, and rectal administration are also contemplated herein.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum that melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases can beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories can be prepared either by thecompressed method or by molding. The typical weight of a rectalsuppository is about 2 to 3 gm.

Tablets and capsules for rectal administration are manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration.

Formulations suitable for transdermal administration can be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound as an optionally buffered aqueoussolution of, for example, 0.1 to 0.2 M concentration with respect to theactive compound. Formulations suitable for transdermal administrationcan also be delivered by iontophoresis (see e.g., PharmaceuticalResearch 3 (6): 318 (1986)) and typically take the form of an optionallybuffered aqueous solution of the active compound.

The pharmaceutical compositions can also be administered by controlledrelease means and/or delivery devices (see e.g., in U.S. Pat. Nos.3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899;4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548;5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566). The activecompounds or pharmaceutically acceptable derivatives can be preparedwith carriers that protect the compound against rapid elimination fromthe body, such as time-release formulations or coatings.

In one embodiment of the compositions and methods provided herein, thetherapeutic agent is administered locally in a slow release deliveryvehicle, for example, encapsulated in a colloidal dispersion system orin polymer stabilized crystals. Useful colloidal dispersion systemsinclude nanocapsules, microspheres, beads, and lipid-based systems,including oil-in-water emulsions, micelles, mixed micelles, andliposomes. For example, the colloidal dispersion system can be aliposome or microsphere. Liposomes are artificial membrane vesicles thatare useful as slow release delivery vehicles when injected or implanted.Some examples of lipid-polymer conjugates and liposomes are disclosed inU.S. Pat. No. 5,631,018, which are incorporated herein by reference inits entirety. Other examples of slow release delivery vehicles arebiodegradable hydrogel matrices (U.S. Pat. No. 5,041,292), dendriticpolymer conjugates (U.S. Pat. No. 5,714,166), and multivesicularliposomes (Depofoam®′ Depotech, San Diego, Calif.) (U.S. Pat. Nos.5,723,147 and 5,766,627). One type of microspheres suitable forencapsulating therapeutic agents for local injection (e.g., intosubdermal tissue) is poly(D,L)lactide microspheres, as described in D.Fletcher, Anesth. Analg. 84:90-94, (1997). For example, a slow releaseformulation containing an effective amount of sHASEGP or a soluble humanhyaluronidase domain thereof, such as 1 to 5000 Units/ml, can beemployed for various uses or to treat various conditions, including, butnot limited to, cosmetic formulations and treatment of spinal cordinjuries.

Desirable blood levels can be maintained by a continuous infusion of theactive agent as ascertained by plasma levels. It should be noted thatthe attending physician would know how to and when to terminate,interrupt or adjust therapy to lower dosage due to toxicity, or bonemarrow, liver or kidney dysfunctions. Conversely, the attendingphysician would also know how to and when to adjust treatment to higherlevels if the clinical response is not adequate (precluding toxic sideeffects).

The efficacy and/or toxicity of the sHASEGP polypeptide and/or itsinhibitor (s), alone or in combination with other agents, such astherapeutically effective agents, also can be assessed by the methodsknown in the art (see, e.g., O & Apos; Reilly, Investigational New Drugs15: 5-13 (1997)).

6. Articles of Manufacture

The sHASEGP polypeptides or soluble human hyaluronidase domains thereofor compositions containing any of the preceding agents can be packagedas articles of manufacture containing packaging material, a compound orsuitable derivative thereof provided herein, which is effective fortreatment of a diseases or disorders contemplated herein, within thepackaging material, and a label that indicates that the compound or asuitable derivative thereof is for treating the diseases or disorderscontemplated herein. The label can optionally include the disorders forwhich the therapy is warranted.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art (see, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,352). Examples of pharmaceuticalpackaging materials include, but are not limited to, blister packs,bottles, tubes, inhalers, pumps, bags, vials, containers, syringes,bottles, and any packaging material suitable for a selected formulationand intended mode of administration and treatment. A wide array offormulations of the compounds and compositions provided herein arecontemplated, as are variety treatments for any disorder in which HCVinfection is implicated as a mediator or contributor to the symptoms orcause.

Kits containing the compositions and/or the combinations withinstructions for administration thereof are also provided herein. Thekit can further include a needle or syringe, typically packaged insterile form, for injecting the complex, and/or a packaged alcohol pad.Instructions are optionally included for administration of the activeagent by a clinician or by the patient. For example, provided herein isa kit containing a small volume syringe with an effective amount ofsHASEGP or a soluble human hyaluronidase domain thereof, such as 1 to5000 Units of the soluble glycoprotein, in a 5 to 50 μl volume,optionally containing a second syringe containing a viscoelastic. Alsoprovided herein is a kit containing a small volume syringe containing aneffective amount of sHASEGP or a soluble human hyaluronidase domainthereof, such as 1 to 500 Units of the soluble glycoprotein, and atherapeutic amount of a second active ingredient, such as a drug, asmall molecule, a protein or a nucleic acid.

K. Animal Models

Transgenic animal models and animals, such as rodents, including miceand rats, cows, chickens, pigs, goats, sheep, monkeys, includinggorillas, and other primates, are provided herein. In particular,transgenic non-human animals that contain heterologous nucleic acidencoding a sHASEGP polypeptide or a transgenic animal in whichexpression of the polypeptide has been altered, such as by replacing ormodifying the promoter region or other regulatory region of theendogenous gene are provided. Such an animal can by produced bypromoting recombination between endogenous nucleic acid and an exogenoussHASEGP gene that could be over-expressed or mis-expressed, such as byexpression under a strong promoter, via homologous or otherrecombination event.

Transgenic animals can be produced by introducing the nucleic acid usingany know method of delivery, including, but not limited to,microinjection, lipofection and other modes of gene delivery into agermline cell or somatic cells, such as an embryonic stem cell.Typically the nucleic acid is introduced into a cell, such as anembryonic stem cell (ES), followed by injecting the ES cells into ablastocyst, and implanting the blastocyst into a foster mother, which isfollowed by the birth of a transgenic animal. Generally introduction ofa heterologous nucleic acid molecule into a chromosome of the animaloccurs by a recombination between the heterologous sHASEGP-encodingnucleic acid and endogenous nucleic acid. The heterologous nucleic acidcan be targeted to a specific chromosome. In some instances, knockoutanimals can be produced. Such an animal can be initially produced bypromoting homologous recombination between a sHASEGP polypeptide gene inits chromosome and an exogenous sHASEGP polypeptide gene that has beenrendered biologically inactive (typically by insertion of a heterologoussequence, e.g., an antibiotic resistance gene). In one embodiment, thishomologous recombination is performed by transforming embryo-derivedstem (ES) cells with a vector containing the insertionally inactivatedsHASEGP polypeptide gene, such that homologous recombination occurs,followed by injecting the ES cells into a blastocyst, and implanting theblastocyst into a foster mother, followed by the birth of the chimericanimal (“knockout animal”) in which a sHASEGP polypeptide gene has beeninactivated (see Capecchi, Science 244: 1288-1292 (1989)). The chimericanimal can be bred to produce homozygous knockout animals, which canthen be used to produce additional knockout animals. Knockout animalsinclude, but are not limited to, mice, hamsters, sheep, pigs, cattle,and other non-human mammals. For example, a knockout mouse is produced.The resulting animals can serve as models of specific diseases, such ascancers, that exhibit under-expression of a sHASEGP polypeptide. Suchknockout animals can be used as animal models of such diseases e.g., toscreen for or test molecules for the ability to treat or prevent suchdiseases or disorders.

Other types of transgenic animals also can be produced, including thosethat over-express the sHASEGP polypeptide. Such animals include“knock-in” animals that are animals in which the normal gene is replacedby a variant, such as a mutant, an over-expressed form, or other form.For example, one species', such as a rodent's endogenous gene can bereplaced by the gene from another species, such as from a human. Animalsalso can be produced by non-homologous recombination into other sites ina chromosome; including animals that have a plurality of integrationevents.

After production of the first generation transgenic animal, a chimericanimal can be bred to produce additional animals with over-expressed ormis-expressed sHASEGP polypeptides. Such animals include, but are notlimited to, mice, hamsters, sheep, pigs, cattle and other non-humanmammals. The resulting animals can serve as models of specific diseases,such as cancers, that are exhibit over-expression or mis-expression of asHASEGP polypeptide. Such animals can be used as animal models of suchdiseases e.g., to screen for or test molecules for the ability to treator prevent such diseases or disorders. In a specific embodiment, a mousewith over-expressed or mis-expressed sHASEGP polypeptide is produced.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

L. Therapeutic Uses of sHASEGP

Naturally occurring hyaluronidase enzymes from slaughterhouses have beenthe principle source of clinical enzyme preparations for over fortyyears. Bovine and Ovine testicles are the principle source of thismaterial. These clinical enzyme preparations however are very crude,sold in preparations ranging from 0.5-5% purity based upon knownspecific activities between 30-100,000 Units/mg. Thus their lack ofpurity combined with their slaughterhouse origin, leave them as bothimmunogenic to humans and as potential source of Jacob Creutzfelddisease and other bovine and ovine pathogens. Anaphylactic reactions tobovine and ovine hyaluronidase preparations are known to occur.

Cattle or bacterially derived hyaluronidase have been used in thetreatment of diseases associated with excess hyaluronic acid and toenhance the circulation of physiological fluids and/or therapeuticagents. For example, bovine hyaluronidase can be co injected withanesthesia in peribulbar, retrobulbar and sub-Tenon's blocks forophthalmic surgical procedures. Moreover, increased surgicalcomplications occur in its absence (Brown S M et al. J Cataract RefractSurg. 1999 September; 25(9): 1245-9.). Bovine hyaluronidase is also usedas an antidote to local necrosis from paravenous injection of necroticsubstances such as vinka alkaloids (Few, B. J. (1987) Amer. J. Matern.Child Nurs. 12, 23-26). Bovine testes hyaluronidase is also useful forthe treatment of ganglion cysts (Paul et al. J Hand Surg 1997 April; 22(2): 219-21). Hyaluronidase can also be used to facilitate subcutaneousdelivery of fluids in hypodermoclysis (Berger E Y, Am Geriatr Soc 1984March; 32(3):199-203). Hyaluronidase has also been utilized to reduceintraocular pressure in the eyes of glaucoma patients and cataractpatients receiving viscoelastics (U.S. Pat. No. 4,820,516 issued Apr.11, 1989).

Cattle or bacterially derived hyaluronidases have also been used as a“spreading agent” to enhance the activity of chemotherapeutics and/orthe accessibility of tumors to chemotherapeutics (Schuller et al., 1991,Proc. Amer. Assoc. Cancer Res. 32:173, abstract no. 1034; Czejka et al.,1990, Pharmazie 45:H.9). Combination chemotherapy with hyaluronidase iseffective in the treatment of a variety of cancers including urinarybladder cancer (Horn et al., 1985, J. Surg. Oncol. 28:304-307), squamouscell carcinoma (Kohno et al., 94, J. Cancer Res. Oncol. 120:293-297),breast cancer (Beckenlehner et al., 1992, J. Cancer Res. Oncol.118:591-596), and gastrointestinal cancer (Scheithauer et al., 1988,Anticancer Res. 8:391-396). Hyaluronidase is effective as the soletherapeutic agent in the treatment of brain cancer (gliomas) (PCTpublished application no. WO88/02261, published Apr. 7, 1988).Administration of hyaluronidase also induces responsiveness ofpreviously chemotherapy-resistant tumors of the pancreas, stomach,colon, ovaries, and breast (Baumgartner et al., 1988, Reg. Cancer Treat.1:55-58; Zanker et al., 1986, Proc. Amer. Assoc. Cancer Res. 27:390).Unfortunately, the contaminants and non human nature of suchhyaluronidases result in anaphylactic reactions.

In addition to its indirect anticancer effects, cattle derivedhyaluronidase has direct anticarcinogenic effects. Hyaluronidaseprevents growth of tumors transplanted into mice (De Maeyer et al.,1992, Int. J. Cancer 51:657-660) and inhibits tumor formation uponexposure to carcinogens (Pawlowski et al., 1979, Int. J. Cancer23:105-109; Haberman et al., 1981, Proceedings of the 17th AnnualMeeting of the American Society of Clinical Oncology, Washington, D.C.,22:105, abstract no. 415).

Given the value of cattle-derived hyaluronidases as a therapeutic,particularly in chemotherapy in conjunction with conventionalchemotherapeutics or as a chemotherapeutic in and of itself, there is aneed in the field for substantially pure preparations of hyaluronidaseof human origin. There is also a need for efficient, cost-effectivemethods of making hyaluronidase to provide commercially significantquantities of the enzyme. The present invention addresses theseproblems.

Hyaluronic acid is an essential component of the extracellular matrix.Hyaluronic acid is found in the connective tissue of mammals and is themain constituent of the vitreous of the eye. In connective tissue, thewater of hydration associated with hyaluronic acid creates spacesbetween tissues, thus creating an environment conducive to cell movementand proliferation. Hyaluronic acid plays a key role in biologicalphenomena associated with cell motility including rapid development,regeneration, repair, embryogenesis, embryological development, woundhealing, angiogenesis, and tumorigenesis (Toole, 1991, Cell Biol.Extracell. Matrix, Hay (ed), Plenum Press, New York, 1384-1386; Bertrandet al., 1992, Int. J. Cancer 52:1-6; Knudson et al., 1993, FASEB J.7:1233-1241). In addition, hyaluronic acid levels correlate with tumoraggressiveness (Ozello et al., 1960, Cancer Res. 20:600-604; Takeuchi etal., 1976, Cancer Res. 36:2133-2139; Kimata et al., 1983, Cancer Res.43:1347-1354).

Following spinal cord injury, glial scars are produced by astrocytes andcontain chondroitin sulfate proteoglycans (CSPGs). CSPGs play a crucialrole in the inhibition of axon growth (Levine, 1994; Powell et al.,1997). For example, during fetal development, CSPGs repel axons andinhibit neural cell adhesion. CSPG's also play an important role inboundary formation (Snow et al., 1990, 1992; Powell and Geller, 1999).In addition the expression of CSPG increases following injury of CNS(Mckeon et al., 1991; Davies et al., 1997).

Studies indicate that the inhibitory effects of CSPGs are principallydue to the chondroitin sulfate (CS) glycosaminoglycan (GAG) sugar chain(Snow et al., 1990; Cole and McCable, 1991; Geisert and Bidanset, 1993).This is supported by the finding that administration of bacterialchondroitinase in fact promote axon regeneration when administeredintrathecally. Moreover, electrophysiological experiments determinedthat regenerated CST axons established functional connections (Bradburyet al., 2002). In addition to their direct inhibitory effects, CSPGscould also interact with cell adhesion molecules or neurotrophic factorsto influence neurite outgrowth (Roberts et al., 1988; Ruoslahti andYamaguchi, 1991; Milev et al., 1994). Recombinant mammalianHyaluronidases are thus useful to reverse the inhibition of CSPG's inthe glial scar and to promote axon regeneration following injury.

The amount of sHASEGP required to sufficiently degrade CSPG's in theglial scar will vary. In some cases repeated administration of 10-5000Units of sHASEGP by intrathecal delivery will be required to remove theCSPG's in the scar. In other cases, sustained release of sHASEGP throughuse of a slow release formulation may be preferred. Alternatively,administration of gene therapy vectors encoding sHASEGP may be effectiveto enhance clearance of CSPG's.

sHASEGPs can also be utilized for the treatment of herniated disks in aprocess known as chemonucleolysis. Chondroitinase ABC, and enzymecleaving similar substrates as sHASEGP can induce the reduction ofintradiscal pressure in the lumbar spine. (Sasaki et al., 2001, Ishikawaet al., 1999). There are three types of disk injuries. A protruded diskis one that is intact but bulging. In an extruded disk, the fibrouswrapper has torn and the NP has oozed out, but is still connected to thedisk. In a sequestered disk, a fragment of the NP has broken loose fromthe disk and is free in the spinal canal. Chemonucleolysis is effectiveon protruded and extruded disks, but not on sequestered disk injuries.In the United States, chemonucleolysis is approved only for use in thelumbar (lower) spine. In other countries, it has also been usedsuccessfully to treat cervical (upper spine) hernias. Chemonucleolysisis thus a conservative alternative to disk surgery when it is preferableto reduce disk pressure.

The precise composition and structure of the carbohydrate chain(s) on aglycoprotein can directly influence its serum lifetime, since cells inthe liver and reticulo-endothelial system can bind and internalizecirculating glycoproteins with specific carbohydrates. Hepatocytes havereceptors on their surfaces that recognize oligosaccharide chains withterminal (i.e., at the outermost end(s) of glycans relative to thepolypeptide) Gal residues, macrophages contain receptors for terminalMan or GlcNAc residues, and hepatocytes and lymphocytes have receptorsfor exposed fucose residues. No sialic acid-specific receptors have beenfound, however. Although somewhat dependent on the spatial arrangementof the oligosaccharides, as a general rule, the greater the number ofexposed sugar residues recognized by cell surface receptors in the liverand reticulo-endothelial system, the more rapidly a glycoprotein will becleared from the serum. Because of the absence of sialic acid-specificreceptors, however, oligosaccharides with all branches terminated, or“capped,” with sialic acid will not promote the clearance of the proteinto which they are attached.

The presence and nature of the oligosaccharide chain(s) on aglycoprotein can also affect important biochemical properties inaddition to its recognition by sugar-specific receptors on liver andreticulo-endothelial cells. Removal of the carbohydrate from aglycoprotein will usually decrease its solubility, and it may alsoincrease its susceptibility to proteolytic degradation by destabilizingthe correct polypeptide folding pattern and/or unmaskingprotease-sensitive sites. For similar reasons, the glycosylation statusof a protein can affect its recognition by the immune system.

sHASEGPs can be used to remove the cumulus cells surrounding an eggprior to cryopreservation and other In Vitro fertilization techniquessuch an intracytoplasmic sperm injection (ICSI). Hyaluronidase can beadded to harvested oocytes between 10-200 U/ml in buffered saltsolutions. Oocytes are separated from the released cumulus cells throughaspiration and washed through several washes with media lackinghyaluronidase. The eggs can then be processed for cryopreservation orIVF techniques.

sHASEGPs are also useful for the more effective penetration ofchemotherapeutic agents into solid tumors. sHASEGPs can be injectedintratumorally with anti-cancer agents or intravenously for disseminatedcancers or hard to reach tumors. The anticancer agent can be achemotherapeutic, an antibody, a peptide, or a gene therapy vector,virus or DNA. Additionally, sHASEGP's can be used to recruit tumor cellsinto the cycling pool for sensitization in previously chemorefractorytumors that have acquired multicultural drug resistance St Croix et alCancer Lett 1998 Sep. 11; 131(1): 35-44). sHASEGPs are also useful toenhance delivery of biologics such as monoclonal antibodies, cytokinesand other drugs to tumors that accumulate glycosaminoglycans. Manytumors delete genes involved with the catabolism of glycosaminoglycanssuch that localized accumulation can prevent antineoplastic agents andthe immune system from reaching the tumor mass.

sHASEGP can also be used to increase the sensitivity of tumors that areresistant to conventional chemotherapy. In one embodiment, sHASEGP isadministered to a patient having a tumor associated with a LuCa-1 defectin an amount effective to increase diffusion around the tumor site(e.g., to increase circulation of chemotherapeutic factors (e.g., tofacilitate circulation and/or concentrations of chemotherapeutic agentsin and around the tumor site), inhibit tumor cell motility (e.g., by HAdegradation) and/or to lower the tumor cell(s) threshold of apoptosis(i.e., bring the tumor cell(s) to a state of anoikis), a state thatrenders the tumor cell(s) more susceptible to the action ofchemotherapeutic agents or other agents that may facilitate cell death,preferably preferentially facilitate programmed cell death of cells inanoikis. Chemotherapeutic agents as used herein is meant to encompassall molecules, synthetic (e.g., cisplatin) as well as naturallyoccurring (e.g., tumor necrosis factor IF)), that facilitate inhibitionof tumor cell growth, and preferably facilitate, more preferablypreferentially facilitate tumor cell death.

Of particular interest is the use of sHASEGP for the treatment ofmetastatic and non-metastatic cancers, particularly metastatic cancers,having decreased to undetectable hyaluronidase activity relative tonon-cancerous (normal) cells. sHASEGP can be used as a chemotherapeuticagent (alone or in combination with other chemotherapeutics) in thetreatment of any of a variety of cancers, particularly invasive tumors.For example, sHASEGP can be used in the treatment of small lung cellcarcinoma, squamous lung cell carcinoma, as well as cancers of thebreast, ovaries, head and neck, or any other cancer associated withdepressed levels of hyaluronidase or with a defective LuCa-1 (hpHAse)gene (e.g., a LuCa-1 gene that does not provide for expression ofadequate hpHAse levels or encodes a defective hpHAse that does notprovide for an adequate level of hyaluronidase activity) or other defectassociated with decreased hyaluronan catabolism. sHASEGP is preferablefor the treatment of malignancies associated with deficient HAcatabolism as it does not require cellular participation for degradationto occur.

The specific dosage appropriate for administration can be readilydetermined by one of ordinary skill in the art according to the factorsdiscussed above (see, for example, Harrison's Principles of InternalMedicine, 11th Ed., 1987). In addition, the estimates for appropriatedosages in humans may be extrapolated from determinations of the levelof enzymatic activity of sHASEGP in vitro and/or dosages effective inanimal studies. For example, 70-300 TRU hyaluronidase is effective inreducing the tumor load in a scid mouse. Given this information, thecorresponding dosages in the average 70 kg human would range from about250,000-1,200,000 TRU hyaluronidase. The amount of sHASEGP administeredto a human patient is generally in the range of 1 TRU to 5,000,000 TRUof enzymatic activity, preferably between about 1,000 TRU to 2,500,000TRU, more preferably between about 100,000 TRU to 1,500,000 TRU,normally between about 250,000 TRU and 1,200,000 TRU, with about 725,000TRU representing average prescribed doses.

In one embodiment, a sHASEGP is formulated in a 0.15 M saline solutioncontaining sHASEGP at a concentration of about 150,000 TRU/cc. Theformulation is then injected intravenously at 15,000 TRU/kg body weightof the patient. Alternatively, the enzyme formulation may also beinjected subcutaneously to allow the hyaluronidase to perfuse around thetumor site. In a preferred embodiment, sHASEGP is injected peritumorallyor into the tumor mass. In another preferred embodiment, sHASEGP isformulated as a liposome and is delivered by injection eitherintravenously or at or near the site of cancerous cells associated witha defect in the LuCa-1 (hpHAse) gene. Injection of sHASEGP intravenouslyresults in sHASEGP in the tumor site. Moreover, Super Sialated sHASEGPis preferably for parenteral administration in that the terminal sialicacids on sHASEGP prevent the clearance of the enzyme from circulation bythe reticuloendothelial system. Comparisons of super sialated sHASEGP tonon-sialated bovine and ovine hyaluronidases reveal that substantiallymore favorable pharmacokinetics is achieved.

Facilitation of Gene Therapy

The efficacy of most gene delivery vehicles in vivo does not correspondto the efficacy found in vitro. Glycosaminoglycans can hinder thetransfer and diffusion of DNA and viral vectors into many cell types.The levels such extracellular matrix material can hinder the processconsiderably. Dubensky et al., (Proc Natl Acad Sci USA 1984 December;81(23):7529-33) demonstrated that hyaluronidase when combined withcollagenase could facilitate transduction of DNA in vivo. It has beendemonstrated that adeno associated virus is also amenable tohyaluronidase mediated gene therapy Favre et al., (Gene Ther 2000August; 7(16):1417-20).

We have determined herein that channels of defined size in theextracellular matrix are opened with sHASEGP. These pores do not enhancethe diffusion of substances greater than about 200-500 nm in diameter.However, smaller molecules such as retroviruses, adenoviruses,adeno-associated viruses and DNA complexes are amenable to sHASEGPmediated diffusion.

Alternatively, viruses can be armed with the sHASEGP gene to facilitatetheir replication and spread within a target tissue for example. Thetarget tissue can be a cancerous tissue whereby the virus is capable ofselective replication within the tumor. The virus can also be anon-lytic virus wherein the virus selectively replicates under a tissuespecific promoter. As the viruses replicate, the coexpression of sHASEGPwith viral genes will facilitate the spread of the virus in vivo.

Alternatively the nucleic acid of interest and a sHASEGP, can be usedsimultaneously or consecutively or so as to be staggered over time.Simultaneously refers to a coadministration. In this case, these twoessential components can be mixed to form a composition prior to beingadministered, or can be administered at the same time to the cell or thehost organism. It is also possible to administer them consecutively,that is to say one after the other, irrespective of which component ofthe combination product according to the invention is administeredfirst. Finally, it is possible to use a mode of administration which isstaggered over time or is intermittent and which stops and restarts atintervals which may or may not be regular. It is pointed out that theroutes and sites of administration of the two components can bedifferent. According to one particularly preferred embodiment, thesHASEGP is administered before the nucleic acid, with the route ofadministration of the two components preferably being similar. The timeinterval between the injections is not critical and can be defined bythe skilled person. It is possible to recommend an interval of from 10min to 72 h, advantageously of from 30 min to 48 h, preferably of from 1to 24 h and, very preferably, of from 1 to 6 h.

In addition, the combination product according to the invention can alsobe combined with one or more molecule(s) which is/are intended toimprove the nucleic acid administration. The molecules can be moleculeswhich have a protective effect on the nucleic acid (protection withregard to degradation in the cell), which improve its penetration or itsexpression in the host cell (fusogenic peptide, nuclear localizationsignal, etc.), which enable one particular cell type to be targeted(ligand or antibody which recognizes a cell surface protein, etc.), orwhich prolong the therapeutic effect (immunosuppressive agent, etc.).The combination product can also be combined with agents that facilitatetransfection (proteins, etc.).

The combination product according to the invention can be prepared witha view to local or parenteral administration or to administration by thedigestive route. Routes which may in particular be mentioned are theintragastric, subcutaneous, intracardiac, intravenous, intraperitoneal,intrasynovial, intratumor, intrapulmonary, intranasal and intratrachealroutes, and, very particularly, the intramuscular route. Theadministration can be effected by means of any technique of the art(injection, oral route, aerosol, instillation, etc.), as a single doseor as a dose that is repeated once or several times after a particulartime interval. The route of administration can be adjusted to suit thegene of interest to be transferred and the disease to be treated. Theformulation can include pharmaceutically acceptable vehicles(excipients, adjuvants, etc.). The substance leading to disorganizationof the extracellular matrix and the nucleic acid of interest arepreferably dissolved in a buffer which is suitable for pharmaceuticaluse and which can be hypertonic, hypotonic or isotonic. Various bufferscan be envisaged. Those which may be mentioned by way of illustrationare a physiological saline solution (0.9% NaCl), a nonphysiologicalsaline solution (1.8% NaCl), a Hepes-Ringer solution, a Lactate-Ringersolution, a buffer which is based on Tris-HCl (10 mM Tris-HCl, pH 7.5 to8, 1 mM EDTA; 10 mM Tris-HCl, pH 7.5 to 8, 1 mM MgCl.sub.2), a phosphatebuffer (Krebs phosphate H.sub.2 O buffer), a sugar (glucose, sucrose,trehalose, etc.) solution, or simply water.

Hypodermoclysis

Hypodermoclysis, the subcutaneous infusion of fluids, is a useful andeasy hydration technique suitable for mildly to moderately dehydratedadult patients, especially the elderly. The method is considered safeand does not pose any serious complications. The most frequent adverseeffect is mild subcutaneous edema that can be treated by local massageor systemic diuretics. Approximately 3 L can be given in a 24-hourperiod at two separate sites. Common infusion sites are the chest,abdomen, thighs and upper arms. The preferred solution is normal saline,but other solutions, such as half-normal saline, glucose with saline or5 percent glucose, can also be used. Potassium chloride can be added tothe solution bag if needed. Additionally, other drugs can be deliveredthrough similar routes. Human sHASEGP can be added to enhance fluidabsorption and increase the overall rate of administration. HumansHASEGP is preferable for repeated Hypodermoclysis over slaughterhouse-derived enzymes in that it not likely to be immunogenic as thebovine enzyme is known to be. It may be administered at home by familymembers or a nurse; the technique should be familiar to every familyphysician.

In ambulatory patients, hypodermoclysis sites include the abdomen, upperchest, above the breast, over an intercostal space and the scapulararea. In bedridden patients, preferred sites are the thighs, the abdomenand the outer aspect of the upper arm. After one to four days, theneedle and tubing should be changed, although infusion sets have beenleft in place for much longer periods without complications.Administration of 500-mL boluses over one or two hours three times a daycan also be given, with 150 U of sHASEGP given at the subcutaneous sitebefore the first morning infusion

Facilitation of Therapeutic Injections.

Many molecules injected percutaneously reach circulation slowly or withvery low efficiency. Several factors regulate the pharmacokinetics andpharmacodynamics of molecules injected subcutaneously (SC) orintramuscularly (IM). Generally, larger molecules reach circulation moreslowly and less efficiently without active transport into circulation.Subcutaneous bioavailability is determined by calculating the ratio ofarea under the curves for SC verses intravenous administration(AUC_(SC)/AUC_(intravenous)). A second factor is charge and affinity formatrix molecules that may play a role in sequestration of moleculessubcutaneously. If these materials are degraded locally they may neverreach their desired targets and thus demonstrate a decreased overallsystemic bioavailability to the target organs.

Large proteins are normally given intravenously so the medicamentdirectly available in the blood stream. It would however be advantageousif a medicament could be given subcutaneously, intramuscularly orintradermally as these administration forms are much easier to handlefor the patient. Especially, if the medicament must be taken regularlyduring the whole life and treatment is to start early, already when thepatient is a child. However, a medicament with a very large and labilemolecule, such as coagulations factor VIII of 170 to 300 kDa, havenormally a very low bioavailability if given subcutaneously,intramuscularly or intradermally, since the uptake is not enough anddegradation is severe.

In addition to the need to increase bioavailability of manysubcutaneously administered biologics, more rapid pharmacokinetics isalso critically important in instances of emergency medicine. The timerequired to reach intravenous access in many patients can prevent anotherwise rapid acting drug when administered systemically from beingutilized. In some cases failure to reach intravenous access is thenfollowed by subcutaneous injection, which leads to additional delay inreaching the target organs. Thus, the more rapid availability ofsubcutaneous drugs would be of benefit as a first line of treatmentrather than to risk the time required to achieve intravenous access.Examples of molecules that can be delivered subcutaneously as well asintravenously include epinephrine, atropine, narcan, lignocaine, anddextrose.

Many molecules injected percutaneously reach circulation slowly or withvery low efficiency. Several factors regulate the pharmacokinetics andpharmacodynamics of molecules injected subcutaneously (SC) orintramuscularly (IM). Generally, larger molecules reach circulation moreslowly and less efficiently without active transport into circulation.Subcutaneous bioavailability is determined by calculating the ratio ofarea under the curves for SC verses intravenous administration(AUC_(SC)/AUC_(intravenous)). A second factor is charge and affinity formatrix molecules that may play a role in sequestration of moleculessubcutaneously. If these materials are degraded locally they may neverreach their desired targets and thus demonstrate a decreased overallsystemic bioavailability to the target organs.

Large proteins are normally given intravenously so the medicamentdirectly available in the blood stream. It would however be advantageousif a medicament could be given subcutaneously, intramuscularly orintradermally as these administration forms are much easier to handlefor the patient. Especially, if the medicament must be taken regularlyduring the whole life and treatment is to start early, already when thepatient is a child. However, a medicament with a very large and labilemolecule, such as coagulations factor VIII of 170 to 300 kDa, havenormally a very low bioavailability if given subcutaneously,intramuscularly or intradermally, since the uptake is not enough anddegradation is severe.

In addition to the need to increase bioavailability of manysubcutaneously administered biologics, more rapid pharmacokinetics isalso critically important in instances of emergency medicine. The timerequired to reach intravenous access in many patients can prevent anotherwise rapid acting drug when administered systemically from beingutilized. In some cases failure to reach intravenous access is thenfollowed by subcutaneous injection, which leads to additional delay inreaching the target organs. Thus, the more rapid availability ofsubcutaneous drugs would be of benefit as a first line of treatmentrather than to risk the time required to achieve intravenous access.Examples of molecules that can be delivered subcutaneously as well asintravenously include epinephrine, atropine, narcan, lignocaine, anddextrose.

An additional benefit of the invention lies in the ability to deliverequivalent or larger volumes of solutions SC or IM without the pain andmorbidity associated with the pressure and volume of the solution at thesite of injection.

Vitreous Hemorrhage

In an effort to minimize the potential for causing further detachment ortearing of the retina during performance of vitrectomy, it haspreviously been proposed in U.S. Pat. No. 5,292,509 (Hageman), to injectcertain protease-free glycosaminoglycanase enzymes into the vitreousbody, to cause the vitreous body to become uncoupled or “disinserted”from the retina, prior to removal of the vitreous body. Suchdisinsertion or uncoupling of the vitreous body is purported to minimizethe likelihood that further tearing or detachment of the retina willoccur as the vitreous body is removed. Examples of specificprotease-free glycosaminoglycanase enzymes which may be used to bringabout this vitreal disinsertion purportedly include; chondroitinase ABC,chondroitinase AC, chondroitinase B, chondroitin 4-sulfatase,chondroitin 6-sulfatase, hyaluronidase and beta-glucuronidase.

Although hyaluronidase enzyme has been known to be usable for variousophthalmic applications, including the vitrectomy adjunct applicationdescribed in U.S. Pat. No. 5,292,509 (Hageman), published studies haveindicated that the hyaluronidase enzyme may itself be toxic to theretina and/or other anatomical structures of the eye. See, The Safety ofIntravitreal Hyaluronidase; Gottleib, J. L.; Antoszyk, A. N., Hatchell,D. L. and Soloupis, P., Invest Ophthalmol V is Sci 31:11, 2345-52(1990). Moreover, the used of impure slaughterhouse preparations ofhyaluronidase can cause uveitis or inflammation of the eye. The use ofhuman sHASEGP is thus preferable in both its increased potency, purityand lack of animal origin that can give rise to immunogenic reactionsand antibody mediated neutralization following repeated administration.In another embodiment, a pegylated form of a sHASEGP can be injectedinto the eye. Such a pegylated sHASEGP is not cleared from the vitreousin such a rapid fashion and maintains its activity in the vitreous for alonger period of time.

The ophthalmic toxicity of some hyaluronidase preparations has beenconfirmed by other investigators, who have proposed that suchhyaluronidase preparations be used as a toxic irritant for causingexperimentally induced neovascularization of the eye, in animal toxicitymodels, (see An Experimental Model of Preretinal Neovascularization inthe Rabbit; Antoszyk, A. N., Gottleib, J. L., Casey, R. C., Hatchell, D.L. and Machemer, R., Invest Ophthalmol V is Sci 32:1, 46-51 (1991). Theuse of a highly purified sHASEGP devoid of mercury-based and cattle orbacterially derived contaminants is preferable for intraocularprocedures. Moreover, a recombinant human sHASEGP is preferable overslaughterhouse derived preparations in both purity lack of bovinepathogens and reduced risk of immunogenicity. Most preferably apegylated sHASEGP is envisioned.

An enzymatic method using a human sHASEGP is thus provided for treatingophthalmic disorders of the mammalian eye. In one embodiment of theinvention, said sHASEGP is PEGylated to prolong its residence within thevitreous and prevent localized uptake. Prevention of neovascularization,and the increased rate of clearance from the vitreous of materials toxicto retina, are accomplished by administering an amount of hyaluronidaseeffective to liquefy the vitreous humor of the treated eye withoutcausing toxic damage to the eye. Liquefaction of the vitreous humorincreases the rate of liquid exchange from the vitreal chamber. Thisincrease in exchange removes those materials and conditions whosepresence causes ophthalmologic and retinal damage.

Cosmetic Uses of sHASEGP

It is known that hyaluronidase has the effect of depolymerizing the longmucopolysaccharide chains of the fundamental substance, responsible forthe retention of bound water and of the slowing, by capillarycompression, of the diffusion of organic liquids, which eliminatemetabolic wastes. Such retention of water and wastes associated with fatoverloading of the lipocytes, constitutes classical “pigskin” edema or“orange peel” edema. This depolymerization will therefore cut the longchains of mucopolysaccharides into shorter chains, whence theelimination of the bound water, of wastes, restoration of the venous andlymphatic circulation and disappearance of local edema.

Use of sHASEGP by way of subcutaneous administration is thus preferredfor the removal of glycosaminoglycans involved in the accumulation ofso-called cellulite and to promote lymphatic flow. Human sHASEGP ispreferred for the treatment of cellulite in that it is capable ofremoval of said glycosaminoglycans without the inflammatory componentsof slaughter house derived proteins and is of high purity and is notlikely to be immunogenic. The sHASEGP can be administered throughrepeated subcutaneous injections, through transdermal delivery in theform of ointments or creams or through the use of injectable slowrelease formulations to promote the continual degradation ofglycosaminoglycans and prevent their return.

Organ Transplantation

Hyaluronan has several biological effects, that are in part related toits molecular size (West, D. C., Kumar, S. Exp. Cell. Res. 183, 179-196,1989). The content of hyaluronan in an organ increases in differentconditions of inflammation of that organ. Thus, an increasedconcentration of hyaluronan has been shown in tissue from differentorgans characterized by inflammatory-immunological injury such asalveolitis (Nettelbladt et al., Am Rev Resp Dis 1989; 139: 759-762) andmyocardial infarction (Waldenstrom et al., J Clin Invest 1991; 88(5):1622-1628). Other examples are allograft rejection after a renal(Ha'llgren et al., J Exp Med 1990a; 171: 2063-2076; Wells et al.,Transplantation 1990; 50: 240-243), small bowel (Wallander et al.,Transplant Int 1993; 6: 133-137) or cardiac (Hällgren et al., J ClinInvest 1990b; 85:668-673) transplantation; or a myocardial inflammationof viral origin (Waldenstrdm et al., Eur J Clin Invest 1993; 23:277-282).

The occurrence of interstitial edemas in connection with the grafting ofan organ constitutes a severe problem in the field of transplantationsurgery. As much as 25% of the grafts, will swell to such a degree thatthe function will temporarily be lost. Moreover, in 2-3% of the cases,the swelling causes disruption of the kidney, resulting in a massivehaemorrhage.

SHASEGP may be used to degrade accumulated glycosaminoglycans in anorgan transplant. Removal of such glycosaminoglycans promotes removal ofwater from the graft and thus organ function. Dose ranging from500-10,000 Units/kg may be administered to reduce interstitial pressureas such.

Pathologic Accumulations of Glycosaminoglycans in the Brain

Hyaluronan levels are elevated in a number of cerebrospinal pathologicconditions. Levels of cerebrospinal hyaluronan are normally less than200 ug/L in adults (Laurent et al., Acta Neurol Scand 1996 September;94(3):194-206). These levels can elevate to over 8,000 ug/L in diseasessuch as meningitis, spinal stenosis, head injury and cerebralinfarction. Thus administration of sHASEGP by either intrathecaldelivery or systemic injection of super sialated sHASEGP can be utilizedto degrade critically elevated levels of substrate.

The lack of effective lymphatics in the brain can also lead to lifethreatening edema following head trauma. Hyaluronan accumulation is aresult of increased synthesis by HA synthases, and decreaseddegradation. Accumulation of hyaluronan serves the purposed ofincreasing water content in the damaged tissue to facilitate leukocyteextravasation but can be lethal. Administration of human sHASEGP to apatient suffering from head trauma can thus removal tissue hyaluronanaccumulation and the water associated with it. Human sHASEGP can beadministered intrathecally through a shunt or alternatively, SuperSialated sHASEGP can be administered intravenously to reach the braintissue.

Following and ischemic of the brain as occurs in stroke, the hyaluronancontent increases dramatically due to increased expression of HAsynthases and decreased catabolism. Failure of ion pumps and leakage ofplasma into the interstitium results in fluid retention that if notproperly cleared by the lymphatics, results in tissue necrosis. Somegroups have attempted to prevent interstitial fluid accumulationfollowing ischemia reperfusion by blocking vascular permeability.However, once the fluid has extravasated, preventing vascularpermeability can prevent resolution of edema and exacerbate conditions.

Human sHASEGP can also be used in the treatment of edema associated withbrain tumors, particularly that associated with glioblastoma multiform.The edema associated with brain tumors results from the accumulation ofhyaluronan in the non-cancerous portions of the brain adjacent thetumor. Administration of hyaluronidase to the sites of hyaluronanaccumulation (e.g., by intravenous injection or via a shunt) can relievethe edema associated with such malignancies by degrading the excesshyaluronan at these sites. Thus, hyaluronidase is successful in thetreatment of brain tumors not only in the reduction of the tumor massand inhibition of tumor growth and/or metastasis, but it also is usefulin relieving edema associated with the malignancy. Human sHASEGP can beadministered for treatment of edema in a manner similar to that foradministration of bovine testicular hyaluronidase to treat edema (see,e.g., Sa Earp Arq. Braz. Med. 44:217-20).

Treatment of Glycosaminoglycan Accumulation in Cardiovascular Disease

It has been shown that the administration of hyaluronidase in animalmodels following experimental myocardial infarct can reduce infarct size(Maclean et al., Science 1976 Oct. 8; 194(4261):199-200). The proposedmechanism by which bovine hyaluronidase reduces infarct size in animalsis by reducing hyaluronan accumulation that occurs following ischemiareperfusion. Reduction of infarct size is believed to occur fromincreased lymph drainage and increased tissue oxygenation and reductionof myocardial water content. While reduced infarct size could beobtained in animal models, the benefits were not realized in largerclinical studies in humans. Bovine testes hyaluronidase possesses aremarkably short serum half life of approximately 3 minutes in animalsand man Wolf et. al., J Pharmacol Exp Ther 1982 August; 222(2):331-7.This short half-life is due to the terminal mannose residues that arereadily recognized by the scavenger receptors of the reticuloendothelialsystem. While small animals may benefit from hyaluronidase due to asmaller vascular bed, an enzyme with increased half-life is needed.Super sialated sHASEGP possesses more favorable pharmacokinetics due tosialation for which there is no scavenger receptor. Super sialatedsHASEGP in doses ranging from 100-200,000 Units/kg may be utilized tofacilitate resolution of excess hyaluronan following ischemiareperfusion and to reduce infarct size.

Super sialated sHASEGP may also be used to limit coronary plaques fromarteriosclerosis. Such plaques accumulate glycosaminoglycans and mediatemacrophage and foam cell adhesion Kolodgie et al., Arterioscler ThrombVasc Biol. 2002 Oct. 1; 22(10):1642-8. Administration of Super SialatedsHASEGP can be used to reduce plaque formation. As repeatedadministration of hyaluronidase is contemplated at doses from100-100,000 U/kg, the need to utilize a human recombinant protein withlow risk of immunogenicity and increased half-life will result insuperior reduction of plaques.

Treatment of Peripheral Tissues Necrosis

Tissue necrosis occurs in many diseases due to venous insufficiency. Thelack of sufficient oxygenation is one of the main obstacles for regrowthof the tissue. It has been demonstrated that intra-arterialhyaluronidase treatment significantly improves the clinical picture inpatients with peripheral arterial occlusive disease (Elder et. al.,Lancet (1980) 648-649). sHASEGP can be injected intra-arterially 3-5times a week at doses from 10-200,000 Units.

Enhancement of Anesthesia

Slaughterhouse-derived hyaluronidase is commonly used for peribulbarblock in local anesthesia prior ophthalmic surgery. The presence of theenzyme prevents the need for additional blocks and speeds the time tothe onset of akinesia (loss of eye movement). Peribulbar and sub-Tenon'sblock are the most common applications of hyaluronidase for ophthalmicprocedures. Since the discontinuation of Wydase®, reports of increaseddiplopia and ptosis have been reported with peribulbar block (Brown etal J Cataract Refract Surg 1999; 25:1245-9).

With Wyeth's discontinuance of Wydase®, bovine testes-derivedhyaluronidase material is now supplied by compounding pharmacies.However, there are several concerns with using an extemporaneouslycompounded sterile product. Compounded preparations are not FDA-approvedproducts. As such, the FDA has no control over the quality orconsistency of the manufacturing process.

SHASEGP from 10-500 Units can be mixed directly with 5 ml 2% lidocaine(Xylocaine), 5 ml 0.5% bupivacaine (Marcaine) and optionally withepinephrine 1:200,000. sHASEGP can be used to increase the onset ofakinesia and to remove the need for additional blocks. sHASEGP is alsoideal for akinesia for cosmetic surgery in blepharoplasties and facelifts. sHASEGP can also be utilized following such surgical proceduresto diffuse anti inflammatories and to reduce tissue swelling.

SHASEGP may also be mixed with a buffering solution such as bicarbonateto prevent discomfort during the injection procedure. SHASEGP can alsobe mixed with anesthesia for lacerations to both reduce the total volumeof material required for injection and to reduce pain from swelling oftissue.

Reduction of Intraocular Pressure

A common side effect occurring in postoperative cataract patients is asignificant early, and occasionally prolonged, rise in intraocularpressure. Such a condition is sometimes serious, especially in patientswith glaucomatous optic disc changes. Although the pressure increasetends to be more severe when visco-elastic agents such as hyaluronicacid are injected into the eye during surgery, the intraocular pressurecan become elevated postoperatively even when such agents are notutilized. Furthermore, such a pressure increase can occur even when noadditional medications are used during the surgical procedure. In somecases, it is advantageous to leave a viscoelastic agent in the eye,which often necessitates giving patients large doses of carbonicanhydrase inhibitors. These inhibitors lower the intraocular pressure bydecreasing the formation of aqueous humor, a fluid that is normallysecreted in the eye, by the ciliary body. Current methods for relievingpostoperative pressure increases in the eye include various types of eyedrops such as beta-adrenergic blocking agents, sympathomimetic agents,miotics, alpha II selective agents, carbonic anhydrase inhibitors andprostaglandin agents.

A preferred method of removing the viscoelastic such as hyaluronic acidis by injection of sHASEGP during or immediately following anteriorsegment or posterior segment surgical procedures, although other methodsof administration known in the art are possible as well. It is preferredif the hyaluronic acid and the sHASEGP are administered by injectioninto the anterior chamber during anterior segment ocular surgicalprocedures to allow the hyaluronic acid to act as a spacer during thestart of the surgical procedure. In some cases of cornealtransplantation, the hyaluronic acid and sHASEGP combination may beplaced on the surface of the intraocular structures prior to suturingthe corneal transplant in place. This combination may also be used inposterior segment surgery, such as retina or vitreous surgery.

In some cases, it may be advisable to leave a visco-elastic agent suchas Healon™, Viscoat™, or other space-occupying substances in theanterior chamber of the eye at the conclusion of surgery. This isespecially true in positive pressure rise when the intraocular contentstend to come forward and press against the posterior surface of thecornea. If this occurs in an eye with a synthetic intraocular lens inplace, pressure on the corneal endothelium can cause significant damageto the cells and subsequent corneal swelling and opacification canoccur, which are associated with decreased vision. Typically, if apatient's intraocular pressure is significantly elevated at theconclusion of the operative procedure, it is necessary to give such apatient large doses of carbonic anhydrase inhibitors, as well as topicaleye drops such as beta-blockers and alpha II agonists in order todecrease aqueous formation and/or to increase aqueous outflow. Theseagents all have significant side effects and, in some instances, arecontraindicated in patients with various types of medical conditionssuch as breathing problems, heart disease or high blood pressure.However, the use of sHASEGP in these situations will eliminate thenecessity of giving these patients large doses of such drugs.

Furthermore, there is a significant amount of hyaluronic acid in thetrabecular meshwork. The sHASEGP will break this down and thereforeimprove the outflow of the aqueous through the trabecular meshwork. Thepatient's intraocular pressure will therefore decrease. The combinationof sHASEGP with other anterior chamber agents, such as a methylcellulose(Ocucoat® for example, commercially available from Storz InstrumentCo.), used as spacers and/or protective agents in cataract surgery, willalso be efficacious in preventing significant pressure rises because itwill in effect open the trabecular meshwork and allow more aqueous humordrainage by breaking down a significant amount of the hyaluronic acidpresent in the trabecular meshwork.

Removal of glycosaminoglycans from the trabecular meshwork is alsouseful for the reduction of intraocular pressure in individualssuffering form open angle glaucoma. Human sHASEGP can be administered bysubconjunctuval injection or injection directly in the anterior chamber.

Ganglion Cysts

The ganglion cyst (also known as a wrist cyst, Bible cyst, or dorsaltendon cyst) is the most common soft tissue mass of the hand. It is afluid filled sac that can be felt below the skin. It is usually attachedto a tendon sheath (lining which lubricates the tendon) in the hand orwrist or connected with an underlying joint; however, some have noobvious connection to any structures. These may also occur in the foot.It often occurs when there is a tear in the ligaments overlying thelining of tendons or joints and the lining herniates out of theligamentous defect causing a bump under the skin. Because there is ofteninflammation associated, the inflamed tissue produces a jelly-like fluidthat fills the protruding sac. They may be rock hard due to a highpressure of the mucous like fluid contained within the cyst, and areoften mistaken for a bony prominence.

sHASEGP can be used to ameliorate ganglion cysts. Intralesionalinjection of sHASEGP from 5-1000 Units followed by fine needleaspiration will remove the cyst without the need for surgery.Corticosteroids may be optionally injected as well with the sHASEGP.Additional injection may be required for some patients.

Myxedema

Glycosaminoglycan (GAG) infiltration of the skin is a feature ofhyperthyroidism, hypothyroidism, pretibial myxedema, scleromyxedema, andscleredema. Hyaluronic acid is the main GAG in all the conditions and innormal skin. There is minimal histologic variability of GAG dermaldistribution. The acquired cutaneous mucinoses exhibit similar skin GAGdistribution and biochemical composition. The morphologic differences infibroblastic activity suggest that the mucinoses of scleredema andscleromyxedema represent a local process, whereas the GAG infiltrationof thyroid diseases may have a systemic origin. These disorders may beameliorated with a sHASEGP from both a local and systemic route ofadministration. For chronic therapy, a PEGylated sHASEGP may beenvisioned.

Pulmonary Uses of sHASEGP

Levels of Hyaluronan in broncheoalveolar lavages (BAL) from normalindividuals are generally below 15 ng/ml. However, BAL levels risedramatically in conditions of respiratory distress (Bjermer Br Med J(Clin Res Ed) 1987 Oct. 3; 295(6602):803-6). In ARDS for example,hyaluronan levels can increase to 500 ng/ml whereas in farmers lung, BALlevels can surpass 1000 ng/ml (Hallgren et al Am Rev Respir Dis. 1989March; 139(3):682-7), (Larrson et al Chest. 1992 January;101(1):109-14). The increased hyaluronan in the lung can prevent oxygendiffusion and gas exchange as well as activating neutrophil andmacrophage responses.

Bovine preparations of hyaluronidase are no preferable for the treatmentof such conditions for a number of reasons. First, slaughterhousetestes-derived preparations of hyaluronidase are known to becontaminated with serine proteases such as acrosin. Secondly, theforeign nature of the bovine enzymes increase the probability of ananaphylactic reaction, which could result in death of the patient. Thusa highly purified preparation of recombinant human sHASEGP can bedelivered by either pulmonary or intravenous delivery. Human sHASEGP canalso be administered to patients suffering from other pulmonarycomplications that are associated with elevated glycosaminoglycans or toenhance the delivery of other co delivered molecules to the lung.

The invention will now be described in greater detail by reference tothe following non-limiting examples

Example 1 Microtiter Based Hyaluronidase Assays

The following example provides for a rapid assay for measurement of thehyaluronidase activity of sHASEGP. This assay can be related to the TRU,the IU or NFU through use of a W.H.O. standard preparation ofhyaluronidase.

Biotinylated Hyaluronan Microtiter Assay

The free carboxyl groups on glucuronic acid residues of Hyaluronan arebiotinylated in a one step reaction using biotin-hydrazide (Pierce),Sulfo NHS (Pierce) and 1-Ethyl dimethylaminopropyl-carbodiimide (Sigma).This biotinylated HA substrate is covalently coupled to a 96 wellmicrotiter plate in a second reaction. At the completion of the enzymereaction, residual substrate is detected with an avidin-peroxidasereaction that can be read in a standard ELISA plate reader. As thesubstrate is covalently bound to the microtiter plate, artifacts such aspH-dependent displacement of the biotinylated substrate does not occur.The sensitivity permits rapid measurement of Hyaluronidase activity fromcultured cells and biological samples with an inter-assay variation ofless than 10%.

a. Protocol

Preparation of Biotinylated HA Substrate

One hundred mg of HA (Sigma Chemicals) was dissolved in 0.1 M MES, pH5.0, to a final concentration of 1 mg/ml and allowed to dissolve for atleast 24 hr at 4° C. prior to coupling of biotin. Sulfo-NHS (Pierce;Rockford Ill.) was added to the CS04 MES solution to a finalconcentration of 0.184 mg/ml. Biotin hydrazide (Pierce) was dissolved inDMSO as a stock solution of 100 mM and added to the CS04 solution to afinal concentration of 1 mM. A stock solution of1-ethyl-3-(3-dimethylaminopropyl)carbidodiimide (EDAC) was prepared as a100 mM stock solution in distilled water and added to the HA biotinsolution at a final concentration of 30 mM. This solution was leftstirring overnight at 4° C. Unlinked biotin and EDAC were removed bydialysis against water with 3 changes of 1000× volume of water. Thedialyzed, biotinylated HA (bHA) was aliquoted and stored at −20° C. forup to several months.

Sulfo-NHS was diluted to 0.184 mg/ml in water with the bHA at aconcentration of 0.2 mg/ml and pipetted into 96 well COVALINK-NH plates(NUNC; Placerville N.J.) at 50 μl per well. EDAC was diluted to 0.123mg/ml in water and pipetted into the COVALINK-NH plates with the bHAsolution resulting in a final concentration of 10 μg/well bHA and 6.15μg/well EDAC. The plates were incubated overnight at 4° C. or for 2 hrat 23° C., which gave comparable results. After covalent immobilizationof bCS04 on the microtiter plates, the coupling solution was removed byshaking and the plates were washed 3 times in PBS containing 2M NaCl and50 mM MgSO4 (Buffer A). The plates could be stored at 4° C. for up toone week.

The COVALINK-NH plates with immobilized bHA were equilibrated with 100μl/well assay buffer—either 0.1 M formate, pH 3.7, 0.1 M NaCl, 1% TRITONX-100 detergent, 5 mM saccharolactone for lysosomal Hyaluronidase; or 10mM Hepes PH 7.4 with 1 mM CaCl₂ and 1 mg/ml Human Serum Albumin (ICN)for neutral-active enzymes. A set of standards for the calibration ofenzyme activity against “relative Turbidity Reducing Units” (rTRU's) wasgenerated by diluting bovine testicular hyaluronidase (Sigma Type VI-S)in neutral enzyme buffer from 1.0 to 1×10⁻⁶ rTRU/well and assaying 100μl/well in triplicate. Samples of acid-active Hyaluronidase were dilutedin lysosomal assay buffer from 1:10 to 1:130,000 were pipetted intriplicate at 100 μl/well. For most assays of tissue extracts and humanplasma, a 30 min incubation at 37° C. was sufficient. Positive andnegative control wells (no enzyme or no ABC (see below), respectively)were included in triplicate.

The reaction was terminated by the addition of 200 μl/well of 6MGuanidine HCl followed by three washes of 300 μl/well with PBS, 2 MNaCl, 50 mM MgSO₄, 0.05% TWEEN 20 detergent (Buffer B). An avidin biotincomplex (ABC) kit (Vector Labs; Burlingame Calif.) was prepared in 10 mlof PBS containing 0.1% TWEEN 20 detergent, which was preincubated for 30min at room temperature during the incubation. The ABC solution wasadded (100 μl/well) and incubated for 30 min at room temperature. Theplate was washed five times with Buffer B, then an o-phenylenediamine(OPD) substrate was added at 100 μl/well by dissolving one 10 mg tabletof OPD in 10 ml of 0.1 M citrate-PO₄ buffer, pH 5.3 and adding 7.5 μl of30% H₂0₂. The plate was incubated in the dark for 10-15 min, then readusing a 492 nm filter in an ELISA plate reader (Titertek Multiskan PLUS;ICN) monitored by computer using the Delta Soft II plate reader softwarefrom Biometallics (Princeton N.J.). A standard curve using the bovinetesticular hyaluronidase was generated by a four parameter curve fit ofthe commercial hyaluronidase preparation and unknown samples wereinterpolated through their absorbance at 492 nm.

To analyze pH dependence of Hyaluronidases, purified recombinant sHASEGPand bovine testicular hyaluronidase are used. The pH dependence ofenzyme activity is measured by diluting purified sHASEGP or partiallypurified bovine testicular hyaluronidase to 0.1 rTRU in the followingbuffers: 50 mM formate, pH 3-4.5; 50 mM acetate, pH 5-6; 50 mM MES, pH6-7; or 50 mM HEPES, pH 7-8. Samples are assayed for 30 min at 37° C.and activity was expressed as a percent of maximal activity. NaCl wasnot used in buffers, as it can alter the pH optima of testicularhyaluronidase preparations (Gold, Biochem. J. 205:69-74, 1982; Gacesa etal. Biochem. Soc. Trans. 7:1287-1289, 1979); physiological saltconcentrations (0.15 M) decreased the apparent pH optimum, an effectthat was more pronounced in purified preparations of the testicularenzyme than in the original crude sample.

b. Results

Hyaluronan was biotinylated in a one step reaction usingbiotin-hydrazide and EDAC. By limiting the EDAC, which couples the freecarboxyl groups on HA with biotin hydrazide, only a small fraction ofthe total glucuronic acid residues on HA were labeled. This amount ofEDAC (3×10⁻⁵ M) added to HA (2.8×10⁻³M) results in a maximum of onemolecule of biotin hydrazide coupled per 93 disaccharide units of HA.

A four-parameter curve fit of bovine testicular hyaluronidase standardreactions measured at pH 3.7, and diluted from 1.0 to 1×10 TRU/well, wasprepared. Four parameter curve fits were established from the equationy=((A−D)/(1+(conc/C)^B))+D) where log_(it) y=ln (y′/1−y′),y′=(y−D)/(A−D), B=−b/ln 10 and C=EXP (a/B). The four parameters(A,B,C,D) were calculated with a software program that utilized the 2+2algorithm with linear regression (Rodbard et al., Clin. Chem. 22:350,1976). This curve fit incorporates the sigmoidal aspects the standardcurve. Optimal accuracy for measurement of a sample typically occursfrom 0.001 to 0.01 TRU/well for a 30 min incubation. During a 60 minincubation, 1/1000th of a TRU is detectable. A standard logarithmiccurve also can be utilized over a shorter range of values to establish astandard curve fit.

Example 2 Cloning of sHASEGP cDNA

Nucleic acid encoding Human sHASEGP may be obtained by one skilled inthe art through a number of procedures including, but not limited to,artificial gene synthesis, RT-PCR, and cDNA library hybridization (forexample see, Gmachl et al FEBS 336(3) 1993, Kimmel et al., Proc. Natl.Acad. Sci. USA 90 1993 10071-10075). Alternatively, clones encodinghuman sHASEGP may be obtained from IMAGE, or other suppliers of humangene sequences (Invitrogen Clone ID IOH10647).

The full length human PH20 cDNA was calculated to be 2009 nucleotides inlength and contained an open reading frame of 1530 nucleotides. The 5′UTR is unusually large, which can indicate a retained intron and caninhibit translation by preventing the ribosome from binding to thecorrect initiating methionine codon due to 9 non coding start codons inthe 5′UTR. The protein (Genbank Accession number NP_003108) is predictedto comprise 509 amino acids SEQ ID No. 1 with a calculated molecularmass of 58 kDa.

For sequencing of clones, PCR amplified bands were excised, and elutedwith the Gel Extraction Kit (Qiagen) and cloned into the appropriatevectors with compatible ends after restriction digestion. All sequencingreactions were performed on double stranded DNA with the Taq dye deoxyterminator cycle sequencing kit (Applied Biosystems) according to themanufacturer's instructions, and run on an ABI Prism™ automatedsequencer (Applied Biosystems).

The human PH-20 open reading frame was obtained by amplifying a humantestis cDNA library (Clontech, Palo Alto Calif.) by Polymerase ChainReaction using primers SEQ ID NO 14 and SEQ ID NO 47. PCR products weredigested with NheI and BamHI and cloned into the NheI and BamHI sites ofthe vector IRESpuro2 (Clontech).

Example-4 Isolation of sHASEGP from Human pH20 cDNA

A catalytically active secreted recombinant human sHASEGP expressionvector capable of effective glycosylation in mammalian cells wasgenerated as described below. Other expression constructs with promotersand selection genes for different species such as yeast and insect cellsthat are also capable of generating sHASEGP are contemplated. Positiveselection genes such as Glutamine Synthase or Dihydrofolate Reductase(DHFR) may also be used. The examples given below is not intended torestrict but is rather provided as an example of several plasmidexpression systems that may be used.

In order to construct secreted forms of sHASEGP, truncation mutants thatlack the hydrophobic C terminal end were constructed. Using a GPIcleavage prediction program the GPI anchor cleavage site was locatedaround amino acid position N 483 in the full-length GPI-anchoredprotein. A set of seven nested 3′ primers were used to construct a setof seven truncated deletion mutants lacking predicted GPI anchorstarting at position Y 482 and deleted progressively by one amino acid.These primers were designed to have compatible Nhe1 (5′) and BamH1 (3′)sites to clone the truncation mutants in vector Irespuro2 eitheruntagged with a stop codon in the 3′ primer, or as a C terminus Histagged protein for ease of purification and detection. For examplereverse primers SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10 were usedto generate deletion mutants ending at position Y 482, F 481 and I 480without a 6 His tag. Other mutant primers were generated with the samebase design with the appropriate modifications to include and excludethe particular amino acids. For generating His-tagged variants the sameset of primers are used as for non tagged variants except that primerslack the stop codon in the respective reverse primers, the forwardprimer remaining the same (for His tagged construction refer to primerswith SEQ ID No 19, 20, 21, 22, 23, 24 and 25 which are the reverseprimers without stop codon corresponding to non tagged reverse primersfor their respective constructs). Overlapping primers were used toconstruct a six amino acid spacer followed by hexahistidine within BamH1and Not1 sites in Irespuro2 vector such that His-tagged mutants weregenerated by ligation of the PCR amplified and restriction digestedproducts within the Nhe1 and BamH1 sites in the his tag containingIrespuro2 vector.

To identify whether human sHASEGP could be modified at its carboxyterminus to generate a secreted and neutral active enzyme, a series oftruncations were made from the GPI anchor attachment site to thepredicted “catalytic domain” based upon homology with the bee venomenzyme.

DNA encoding the human sHASEGP full length GPI anchored clone inIRESPuro2 was used as a template to generate the various truncateddeletion mutants. Software modeling programs gave several predictedcleavage sites for the full length polypeptide. One of such predictedsites was at amino acid position N 483 (SEQ ID No. 1). PCR primers weredesigned to successively truncate the protein from N483 to generate sixdeletion mutants starting at Y 482 (lacking N) and ending at E 477(lacking P).

a. Protocol

Generating truncation mutant lacking N483:

The full length GPI anchored sHASEGP clone between Nhe1 and BanHI sitein pIRESPuro2 was used as a template. This template was amplified with5′ primer containing NheI site that starts at starting Methionine of thenative signal peptide at M 1 (SEQ ID No. 14), and a 3′ primer containingBamHI site that ends at Y 482 (SEQ ID No. 8). The PCR product was ran ona 1% agarose gel to resolve and confirm the correct sized amplifiedband, gel purified, restriction digested with NheI and BamHI and clonedinto vector pIRESPuro2 (Clontech) between NheI and BamHI sitesgenerating an expression vector for expressing this truncation mutant ofSHASEGP ending at amino acid position N482 and lacking the GPI anchorwith amino acid sequence (SEQ ID No. 5 for the sequence of the resultingpolypeptide of sHASEGP up to Y 482) and nucleotide sequence (SEQ ID No.48—coding nucleotides for polypeptide in SEQ ID No. 5) as indicated.

Generation of the other truncation mutants lacking Y 482, F 481, I 480,Q 479, and P 478 respectively.

The same strategy was used with the only difference being using theappropriate 3′ primer for each mutant. The respective 3′ primers are asfollows:

3′ primer for sHASEGP mutant that lacks the Y 482-SEQ ID No. 9

3′ primer for mutant that lacks the F 481—SEQ ID No. 10

3′ primer for mutant that lacks the I 480—SEQ ID No. 11

3′ primer for mutant that lacks the Q 479—SEQ ID No. 12

3′ primer for mutant that lacks the P 478—SEQ ID No. 13

Generating further deletion mutants to determine the minimally activedomain of sHASEGP:

Further deletions, in blocks of ten to twenty amino acids were made fromthe 3′ end of innermost neutral pH active truncation mutant of sHASEGP,which is sHASEGP up to E 477. The NheI forward primer SEQ ID No. 14) wasused with an appropriately positioned 3′ primer to PCR amplify adeletion mutant of sHASEGP of the desired length from the carboxyterminal end. For example PCR with primers described in SEQ ID No. 14and SEQ ID No. 26 as the 5′ and 3′ primers respectively was used togenerate the polypeptide in SEQ ID No. 49 when expressed from anexpression construct in IresPuro2 vector. Similarly, PCR with reverse3′primers described in SEQ ID No 27, 28, 29, 30, 31 and 32 were used togenerate deletion mutants ending at amino acid positions A 447, S 430, G413, S 394, A 372, and S 347 respectively of the mature sHASEGP. The PCRproducts in each case were digested with NheI and BamHI enzymes and thedigested product cloned into pIresPuro2 vector between NheI and BamHIsites. A few independent clones in the final expression construct fromeach group were tested for secreted neutral active sHASEGP activity bytransient transfection in CHO cells in CD-CHO serum free media(Invitrogen, CA) and samples drawn at indicated time points for assay.Miniprep DNA prepared from overnight cultures was transfected withGenejuice (Novagen, CA) transfection reagent following manufacturerrecommended protocols. Hyaluronidase activity was measured by microtiterassay as described above.

b. Results

Hyaluronidase activity was measured in sHASEGP truncation mutants toidentify the minimally active domain for secreted neutral activehyaluronidase activity.

AMINO ACID 1 TO: U/ML/24 HRS PH 7.4 347 0.000 372 0.000 394 0.000 4130.000 430 0.000 447 0.000 467 0.089 477 0.567 478 0.692 479 0.750 4800.575 481 0.740 482 0.329 483 0.800 509 0.044

The results showed that all six one amino acid deletion mutants endingat indicated amino acids from Y 482 to E 477 gave higher secretedactivity than GPI anchored sHASEGP.

The results also showed that deletions beyond A 467 eliminated anysecreted activity. Secreted neutral activity from the A 467 clonesdecreased to approximately 10% of that found P 478 or N 483 clones. Itwas therefore concluded that more of the carboxy terminal domain ofhuman sHASEGP was required to create the neutral active hyaluronidasedomain than previously assumed from the bee venom enzyme. The cysteinesin the carboxy terminal domain are thus necessary for neutral activity.A very narrow range spanning approximately 10 amino acids before the GPIcleavage site at N 483 thus defined the minimally active domain.

Example-5 Effects of Signal Peptide Modification on sHASEGP SecretoryActivity

Human sHASEGP possesses an unusually long predicted native leaderpeptide. Additionally, the existence of two adjacent cysteine residuesin the leader peptide may lead to aggregation of polypeptide multimerswithin the endoplasmic reticulum during high level expression andtherefore prevent high level expression of a sHASEGP. A series of moreefficient secretory leader peptides were therefore tested to examine fortheir ability to enhance the targeting of sHASEGP for secretion.

a. Protocol

The Kappa leader peptide was constructed by overlapping primer annealingand extension PCR with primers corresponding to sequences in SEQ ID No37, 38, 39 and 40. The resulting PCR amplified kappa sequence wasamplified with flanking primers containing NheI site in the 5′ end (asdescribed in SEQ ID No. 41) and EcoR1 site at the 3′ end (as describedin SEQ ID No. 42). This allowed cloning the Kappa leader peptide (thepolypeptide sequence is as described in SEQ ID No. 43) in the Litmus 39(NEB) vector between NheI and EcoRI sites. sHASEGP has an internal EcoRIsite; therefore this kappa construct between NheI site and EcoRI sitewas further amplified with a 5′ SpeI primer (as described in SEQ ID No.44) and a 3′ MluI primer (as described in SEQ ID No. 45). sHASEGPwithout GPI anchor ending at P 478 was cut out from pIresPuro2 with NheIand BamHI and cloned into a Litmus 39(NEB) vector within the NheI andBamHI sites of the Litmus39 vector. This resulting sHASEGP-containingLitmus vector was digested with SpeI and MluI restriction enzymes andthe kappa leader construct amplified with SpeI and MluI was cloned intoit. Site directed mutagenesis was performed on this Litmus 39 vectorcontaining both Kappa and sHASEGP sequences to generate the in framefusion of Kappa leader sequence to the mature polypeptide of sHASEGP.Primer pairs corresponding to SEQ ID No. 34 and 35 were used to generatethe kappa leader with the native Asp as the terminal amino acid fused tothe F 38 of sHASEGP (up to P 478) (as described in SEQ ID No. 46 for thepolypeptide sequence of the fusion protein). Other primer paircombinations such as embodied by SEQ ID No. 33 with SEQ ID No. 35 wereused to generate Kappa leader ending at the terminal Asp (D) fused to L36 of SHASEGP, SEQ ID No. 33 with SEQ ID No. 36 were used to generateKappa leader ending at the Gly (G) (before the terminal Asp (D)) fusedto L 36 of SHASEGP, and SEQ ID No. 34 with SEQ ID No. 36 were used togenerate Kappa ending at the Gly (G) (before the terminal Asp (D)) fusedto F 38 of SHASEGP. The Kappa-sHASEGP fusions obtained by site directedmutagenesis were gel purified, digested with enzyme DpnI to digest anycarryover parental DNA, and then digested with NheI and Bam HI andcloned in to the NheI/BamHI digested HisIresPuro2 backbone which has thehis tag (six amino acid spacer followed by six histidines) cloned inbetween BamH1 and Not1 sites in pIRESPuro2 vector. Therefore uponligation we obtain a construct that is NheI-kappa-SHASEGP-BamHI-His inpIresPuro2. Four sets of such construct were obtained that wouldcorrespond to the combinations of G or D at the Kappa leader end and L36 or F 38 at the beginning of mature sHASEGP. A few independent clonesfrom each type of construct were transfected into CHO cells in CD-CHOmedium (Invitrogen, CA) to test whether the presence of kappa secretionleader would promote increased levels of secreted protein as compared tonative secretion leader. Miniprep DNA prepared from overnight cultureswere transfected with Genejuice (Novagen, CA) transfection reagentfollowing manufacturer recommended protocols and samples were drawn fortesting by microtiter assay at indicated time points. Hyaluronidaseactivity was measured by microtiter assay as described above.

Mouse IgG Kappa chain leader peptide sHASEGP fusion constructs weretested to test for higher levels of secreted neutral active sHASEGPactivity.

b. Results

U/ML/24 HOURS HUMAN sHASEGP GENE CONSTRUCT PH 7.4 IgG Kappa LeadersHASEGP AA 38-478 HIS6 3.0257 Native Leader sHASEGP AA 1-478 HIS6 0.4857

The enzyme assay results indicated that the IgG Kappa leader was capableof enhancing secretion of sHASEGP approximately 7 to 8 fold higher thanthe native secretion leader when compared with clones P 478, Y 482 or N483 that lacked such a leader. Other kappa leader constructs withvariations of the leader fusion site from the Asp or the Gly of theKappa leader to L36 or F38 of sHASEGP yielded increased levels ofsecreted neutral active hyaluronidase activity as well. These examplesare intended to expand rather than limit the scope of the invention, asother efficient secretory leader sequences may be utilized with the sametechnology.

Example 6 Generation of a Human sHASEGP Expression Vector

A sHASEGP without an eptiope tag was generated by cloning into abicistronic expression cassette, HZ24 (SEQ ID NO: 51). The HZ24 plasmidvector for expression of sHASEGP comprises a pCI vector backbone(Promega), DNA sequence encoding amino acids 1-482 of human PH20hyaluronidase, an internal ribosomal entry site (IRES) from the ECMVvirus (Clontech), and the mouse dihydrofolate reductase (DHFR) gene. ThepCI vector backbone also includes DNA encoding the Beta-lactamaseresistance gene (AmpR), an f1 origin of replication, a Cytomegalovirusimmediate-early enhancer/promoter region (CMV), a chimeric intron, andan SV40 late polyadenylation signal (SV40). The DNA encoding the sHASEGPconstruct contained a Kozak consensus sequence in the Methionine of thenative signal leader and a stop codon at Tyrosine 482. The resultantconstruct pCI-PH20-IRES-DHFR-SV40pa (HZ-24) results in a single mRNAspecies driven by the CMV promoter that encodes amino acids 1-482 ofPH20 and amino acids 1-187 of the dihydrofolate reductase separated bythe internal ribosomal entry site.

The human PH20 open reading frame was amplified from an Invitrogen ORFclone (IOH10647, Invitrogen, Carlsbad Calif.) with a 5′Primer thatintroduced an NheI site and Kozack consensus sequence before theMethionine of PH20 and a reverse primer that introduced a stop codonfollowing Tyrosine 482 and introduced a BamH1 restriction site. Theresultant PCR product was ligated into the plasmid pIRESpuro2 (Clontech,Palo Alto, Calif.) following digest of the PH20 PCR fragment with NheIand BamH1.

Example-7 Generation of a sHASEGP Expressing Cell Line

Non-transfected DG44 CHO cells growing in GIBCO Modified CD-CHO mediafor DHFR(−) cells, supplemented with 4 mM Glutamine and 18 ml PlurionicF68/L (Gibco), were seeded at 0.5×10⁶ cells/ml in a shaker flask inpreparation for transfection. Cells were grown at 37° C. in 5% CO₂humidified incubator with 120 rpm for shaking. Exponentially growingnon-transfected DG44 CHO cells were tested for viability prior totransfection.

60,000,000 viable cells of the non-transfected DG44 CHO cell culture waspelleted and resuspended to a density of 20,000,000 cells in 0.7 mL of2× transfection buffer (2×HeBS=40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mMKCl, 1.4 mM Na₂HPO₄, 12 mM dextrose). To each aliquot of resuspendedcells, 0.09 mL of the linear HZ24 plasmid (250 ug) was added, and thecell/DNA solutions were transferred into 0.4 cm gap BTX (Gentronics)electroporation cuvettes at room temperature. A negative controlelectroporation was performed with no plasmid DNA mixed with the cells.The cell/plasmid mixes were electroporated with a capacitor discharge of330 V and 960 uF or at 350 V and 960 uF.

The cells were removed from the cuvettes after electroporation andtransferred into 5 mL of Modified CD-CHO media for DHFR(−) cells,supplemented with 4 mM Glutamine and 18 ml Plurionic F68/L (Gibco), andallowed to grow in a well of a E-well tissue culture plate withoutselection for 2 days at 37° C. in 5% CO₂ humidified incubator.

Two days post electroporation, 0.5 mL of tissue culture media wasremoved from each well and tested for presence of hyaluronidaseactivity.

Initial Hyaluronidase Activity of HZ24 Transfected DG44 CHO Cells At 40Hours Post Transfection

Activity Dilution Units/ml Transfection 1 1 to 10 0.25 330 V Transaction2 1 to 10 0.52 350 V Negative 1 to 10 0.015 Control

Cells from transfection 2 (350V), were collected from the tissue culturewell, counted and diluted to 10,000 to 20,000 viable cells per mL. A 0.1mL aliquot of the cell suspension was transferred to each well of five,96 well round bottom tissue culture plates. 0.1 mL of CD-CHO media(GIBCO) containing 4 mM Glutamax-1, and without hypoxanthine andthymidine supplements were added to the wells containing cells (finalvolume 0.2 mL).

Ten clones were identified from the 5 plates grown without methotrexate.

Plate/Well Relative Hyaluronidase ID Activity 1C3 261 2C2 261 3D3 2613E5 243 3C6 174 2G8 103 1B9 304 2D9 273 4D10 302 1E11 242 A1 (+) 333control H12 (−) 0 control

Six HZ24 clones were expanded in culture and transferred into shakerflasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9, 1E11, and4D10 were plated into 96-well round bottom tissue culture plates using atwo-dimensional infinite dilution strategy. Diluted clones were grown ina background of 500 non-transfected DG44 CHO cells per well, to providenecessary growth factors for the initial days in culture. Ten plateswere made per subclone.

Clone 3D3 produced 24 visual subclones. Significant hyaluronidaseactivity was measured in the supernatants from 8 of the 24 subclones(>50 Units/mL), and these 8 subclones were expanded into T-25 tissueculture flasks in the presence of 50 nM methotrexate. Clone 3D3 50 nMwas further expanded in 500 nM methotrexate giving rise to clonesproducing in excess of 1,000 Units/ml in shaker flasks (clone 3D3 5M).

Example 8 Production of sHASEGP

A vial of 3D3 5M was thawed and expanded from T flasks through 1 Lspinner flasks in CHO CDM (Invitrogen, Carslbad Calif.) supplementedwith 100 nM Methotrexate and Glutamax (Invitrogen). Cells weretransferred from spinner flasks to a 5 L bioreactor (Braun) at aninoculation density of 4.0×10E5 viable cells per ml. Paramaters weretemperature setpoint, 37 C, pH 7.2 (starting Setpoint), with DissolvedOxygen Setpoint 25% and an air overlay of 0-100 cc/min. At 168 hrs, 250ml of Feed #1 Medium (CD CHO+50 g/L Glucose) was added. At 216 hours,250 ml of Feed #2 Medium (CD CHO+50 g/L Glucose+10 mM Sodium Butyrate)was added, and at 264 hours 250 ml of Feed #2 Medium was added. Thisprocess resulted in a final productivity of 1600 Units per ml with amaximal cell density of 6 million cells/ml. The addition of sodiumbutyrate was found to dramatically enhance the production of sHASEGP inthe final stages of production.

3D3-5M Growth & sHASEGP Production, 5 L Bioreactor

Run Viable % Vol Hours Cells × 10E5 Viable Units/ml (mL) [Glucose] Feed0 4.4 100 0 4500 547 24 5.7 100 0 4500 536 48 10.1 100 37 4500 501 7217.1 99 62 4500 421 96 28.6 99 118 4500 325 120 28.8 99 240 4500 274 14460.2 100 423 4500 161 168 55 100 478 4500 92 250 ml Feed #1 192 66.6 98512 4750 370 216 55.2 92 610 4750 573 250 ml Feed#2 240 53 88 710 5000573 264 49.8 84 852 5000 474 250 ml Feed #2 288 40 70 985 5250 770 31231 61 1467 5250 773 336 25.4 52 1676 5250 690

Example 9 Purification of sHASEGP

Conditioned media from the 3D3 clone was clarified by depth filtrationand tangential flow diafiltration into 10 mM Hepes pH 7.0. SolubleHASEGP was then purified by sequential chromatography on Q Sepharose(Pharmacia) ion exchange, Phenyl Sepharose (Pharmacia) hydrophobicinteraction chromatography, phenyl boronate (Prometics) andHydroxapatite Chromatography (Biorad, Richmond, Calif.).

SHASEGP bound to Q Sepharose and eluted at 400 mM NaCl in the samebuffer. The eluate was diluted with 2M Ammonium sulfate to a finalconcentration of 500 mM ASO4 and passed through a Phenyl Sepharose (lowsub) column, followed by binding under the same conditions to a phenylboronate resin. The sHASEGP was eluted from the phenyl sepharose resinin Hepes pH 6.9 after washing at pH 9.0 in 50 mM bicine without AS04.The eluate was loaded onto a ceramic hydroxyapatite resin at pH 6.9 in 5mM PO4 1 mM CaCl₂ and eluted with 80 mM PO4 pH 7.4 with 0.1 mM CaCl₂.

The resultant purified sHASEGP possessed a specific activity in excessof 65,000 USP Units/mg protein by way of the microturbidity assay usingthe USP reference standard. Purified sHASEGP eluted as a single peakfrom 24 to 26 minutes from a Pharmacia 5RPC styrene divinylbenzenecolumn with a gradient between 0.1% TFA/H₂O and 0.1% TFA/90%acetonitrile/10% H₂O and resolved as a single broad 61 kDa band by SDSelectrophoresis that reduced to a sharp 51 kDa band upon treatment withPNGASE-F. N-terminal amino acid sequencing revealed that the leaderpeptide had been efficiently removed.

N-terminal Amino Acid Sequence biochemically purified sHASEGP.

Position 1 2 3 4 5 6 7 8 9 10 11 Theoretical Leu Asn Phe Arg Ala Pro ProVal Ile Pro Asn Observed — Asn Phe Arg Ala Pro Pro Val Ile Pro Asn

Example 10 Analysis of DG44 CHO-Derived sHASEGP Glycosylation

Conflicting data exists as to whether sHASEGP's from different speciesrequire glycosylation for their catalytic activity. For example, it isreported that enzymatically active bee venom hyaluronidase can besynthesized in cells that lack glycosylation machinery, i.e. such as E.coli. Moreover, treatment of purified bovine testes hyaluronidase withPNGase did not inactivate enzyme activity (Yamagata et al 1997). Otherstudies report loss of activity following deglycosylation and thatdisulfide bonds are additionally required.

As all such previous tests were made using either crude or partiallypurified preparations however, it was not apparent whether the loss ofactivity was a result of exposure of deglycosylated enzyme tocontaminating proteases in the crude preparations or a direct functionalrelationship between glycosylation and catalytic activity.

a. Protocol

To determine if functional N-linked glycosylation could be introducedinto human sHASEGP using a CHO based expression system under proteinfree conditions, a cDNA encoding human sHASEGP-HIS6 was expressed in CHOcells using an IRESpuro bicistronic cassette in chemically definedmedia. Cells were grown for 72 hours in CHO CDM (Invitrogen/Gibco)followed by concentration and tangential flow diafiltration on aPellicon TFF unit (Millipore) with 30 kDa cutoff membranes. Theconcentrate was exchanged with 10 mM Hepes PH 7.4 50 mM NaCl. Thediafiltrate was then loaded on a DEAE streamline sepharose resin andeluted with a NaCl gradient from 0-1M NaCl on a Pharmacia FPLC resin.Human sHASEGP eluted between 10-30% NaCl. Levels of sHASEGP in columnfractions determined that the majority of enzyme was recovered in the10-30% NaCl gradient. The enzyme from the 10-30% NaCl gradient was thenfurther purified through affinity chromatography on an IMAC resincharged with Ni. Human sHASEGP was eluted from the IMAC resin afterwashing with 10 mM Imidizole with 50 mM Acetate PH 5.0. The protein wasconcentrated and dialyzed against 10 mM Hepes PH 7.4. The highlypurified enzyme was determined to possess a specific activity of 97,000Units/mg protein in the presence of 1 mM Calcium and 1 mg/ml HSA in theELISA-based biotinylated substrate microtiter assay.

To detect changes in protein relative molecular mass, purified humansHASEGP was treated with PNGASE or Neuraminidase overnight followed bygel electrophoresis, electotransfer and western blot analysis with anHRP linked anti His6 monoclonal antibody (Qiagen) and ECL detection.

b. Results

Western blot analysis determined that the human sHASEGP produced in CHOcells was sensitive to PNGASE treatment. The relative molecule mass ofhuman sHASEGP revealed that the protein was highly glycosylated. Uponcomplete overnight digestion with PNGASE, human sHASEGP reduced to asingle species confirming that mild heterogeneity of the undigested bandcould be attributed to N-linked sugar residues. PNGaseF partialdigestion showed a series of intermediates shifting from untreated andprogressive shift with longer treatment. Although bands were somewhatdiffuse on a 7% Gel, at least 6 different intermediate isoforms could bevisualized.

Treatment of sHASEGP with Neuraminidase revealed that CHO cells were infact capable of synthesizing sialated human sHASEGP. Upon treatment withneuraminidase and Western Blot analysis of sHASEGP on 7% Gels, CHOderived Human recombinant sHASEGP revealed an approximately 1-3 kDashift in mobility compared to untreated sHASEGP. This is thus the firstreport of the generation of a substantially sialated human sHASEGP. Thisis very valuable for both stability and to enhance serum half-life of ahuman sHASEGP as native sperm sHASEGP from many species lacks sialationand does not react with sialic acid specific lectins.

FACE Analysis of sHASEGP

Analysis of active sHASEGP oligosaccharides by FACE analysis permitsrapid determination of profiles of catalytically active sHASEGP's.

Protocol

Purified Hyaluronidase from the 3D3 5M clone was evaluated using FACE®N-Linked Oligosaccharide Profiling (Prozyme). Oligosaccharides werecleaved from 128.7 μg of glycoproteins by enzymatic digestion withN-Glycanase (a.k.a PNGase), labeled using the fluorophore ANTS, andseparated by electrophoresis. The relative positions of theoligosaccharide bands were determined by running the sample anddilutions of the sample alongside an oligosaccharide standard ladderwhich designated the migration distance in Degree of Polymerization (DP)units.

Results

The N-Profile for the Hyaluronidase sample consists of ten bands ofwhich six (running concomitant with the oligosaccharide standard bandsG5-G12) had intensities greater than 9%. Furthermore, the band runningalongside the G9 standard was the most intense with intensities of35%-46%.

sHASEGP Oligosaccharide Analysis

sHASEGP Degree of Oligosacharide Polymerization Percent of Total 1 15.641.2 2 13.68 3.4 3 11.61 10.0 4 10.04 10.4 5 8.37 35.4 6 7.32 9.7 7 6.149.0 8 5.57 12.4 9 3.84 2.3 10 3.26 0.5

Example 11 Dependence of sHASEGP N-Linked Glycosylation for EnzymeActivity

a. Protocol

Samples of purified HIS6 sHASEGP were mixed with buffer containingNeuraminidase and PNGASE with and without 50 mm Octylglucoside overnightat 37 C. Oligosaccharides were verified to have been removed by gelshift from Western Blot analysis.

b. Results

SAMPLE U/ML No Rx 22.01 Neuraminidase O/N 50 mM OG 23.57 PNGaseF w/50 mMOG 0.0 PNGaseF without 50 mM OG o/n 10.74

Example-12 Activity of sHASEGP Towards Sulfated and Non-SulfatedGlycosaminoglycans

In addition to the microtiter-based assay using HA, the substratespecificity of sHASEGP towards other glycosaminoglycans or proteoglycanscan be tested using a gel shift assay with purified substrates todetermine the activity of sHASEGP towards other glycosaminoglycans. ManyHyaluronidase assays have been based upon the measurement of thegeneration of new reducing N-acetylamino groups (Bonner and Cantey,Clin. Chim. Acta 13:746-752, 1966), or loss of viscosity (De Salegui etal., Arch. Biochem. Biophys. 121:548-554, 1967) or turbidity (Dorfmanand Ott, J. Biol. Chem. 172:367, 1948). With purified substrates all ofthese methods suffice for determination of the presence or absence ofendoglucosamidic activity.

a. Protocol

GEL SHIFT ASSAY—Purified substrates are mixed with recombinant sHASEGPto test for endoglucosidase activity that give rise to increasedmobility in substrate within the gel. Chondroitin Sulfate A, Aggrecanand D were from Calbiochem. Hyaluronan (Human Umbilical Cord)Chondroitin Sulfate C, Dermatan sulfate, and Heparan-sulfate areobtained from Calbiochem. Human umbilical cord Hyaluronan was obtainedfrom ICN. Each test substrate is diluted to 0.1 mg/ml. 10 ul samples ofpurified sHASEGP or conditioned media from sHASEGP expressing cells aswell as are mixed with 90 ul of test substrate in desired buffer andincubated for 3 hours at 37 C. Following incubation samples areneutralized with sample buffer (Tris EDTA PH 8.0, Bromophenol Blue andglycerol) followed by electrophoresis on 15% polyacrylamide gels.Glycosaminoglycans are detected by staining the gels in 0.5% Alcian Bluein 3% Glacial Acetic Acid overnight followed by destaining in 7% GlacialAcetic Acid. Degradation is determined by comparison substrate mobilityin the presence and absence of enzyme.

b. Results

100 Units of sHASEGP_(HIS6) in 10 ul was incubated with 90 ul 10 mMHepes Buffer with 50 ug/ml Human Serum Albumin for 2 hours at 37 Ccontaining 10 ug of various glycosaminoglycans and proteoglycans.Electrophoretic analysis followed by Alcian blue staining revealedincreased mobility shifts to a single species in Chondroitin Sulfate A,C and D, Aggrecan and Hyaluronan but not Heparan Sulfate nor ChondroitinSulfate B. Whereas the undigested glycosaminoglycans ran as a smear inthe middle of the gel, the digested products showed the majority ofalcian blue stain running at the dye front with a small amount ofmaterial running as an incremental ladder.

Example-13 Effects of Metal Ions on sHASEGP Activation

In addition to the requirement of glycosylation for optimal enzymeactivity, human sHASEGP was found to be activated with cations foroptimal enzyme activity. In the process of purification, sHASEGP wasfound to have a low specific activity following successivechromatography steps. The HIS6tagged sHASEGP was found to have a verylow specific activity when purified to homogeneity from DEAE followed bysuccessive Ni-IMAC purifications. As IMAC resins can chelate metal ions,various metals were added back to sHASEGP to determine the relativeenzyme activity.

a. Protocol

Purified sHASEGP was tested following incubation with 0.1 mM nickel(Ni), Cobalt (Co) Zinc (Zn) Calcium (Ca) and Magnesium (Mg) for 2 hoursat room temperature followed by determination of hyaluronidase activityin microtiter based assay.

b. Results

Metal Salt Additive Neutral Activity U/ml NO ADDITIVES 11.909 100 uM Ni6.0306 100 uM Co 8.972 100 uM Zn 3.7476 100 uM Ca 101.9892

A significant increase in hyaluronidase activity was found followingincubation of sHASEGP with 0.1 mM Calcium or 0.1 mM Magnesium. No suchactivation was found following incubation with other metals. Theaddition of Calcium to sHASEGP increased the specific activity of theenzyme to approximately 97,000 units per mg protein based upon A280measurement. A dose response curve of Calcium and Magnesium metals wasthen tested to determine the optimal concentration of metal ions toenzyme.

mM Divalent Metal [Ca++] [Mg++] 100 1 1.3 10 108 104 1 169 164 0.1 12378 0.01 59 18 0.001 47 13 0.0001 39 13 0.00001 55 15

Activation of sHASEGP was found to occur in the micromolar range.Concentrations above 10 mM were inhibitory for both Calcium andMagnesium. To rule out nonspecific activation of substrate rather thanenzyme, Calcium Chloride in 10 mM Hepes buffer was incubated with theimmobilized biotinylated substrate on the microtiter plate followed bywashing. No activation was found when the enzyme was added to theCalcium preincubated plate that had been washed. The activation was alsotested on phospholipase C released native sHASEGP which revealed asimilar activation with Calcium ruling out an artifact of the carboxyterminus HIS6 epitope tag.

Example 14 Effects of Albumin on the Activity of sHASEGP

It was found that the dilution of recombinant rHUPH20 and otherpreparations of slaughterhouse testes-derived hyaluronidases requiredalbumin in addition to Calcium for optimal activity.

a. Protocol

Human Serum Albumin (ICN) was diluted into 10 mM Hepes buffer withCalcium to determine the effects of albumin protein on enzyme activity.Enzyme assays with sHASEGP and commercial preparations were examinedusing both 1 mM CaCl₂ and 1 mg/ml Human Serum Albumin.

b. Results

Activation of hyaluronidase activity was found at high dilutions in thepresence of albumin. It was not clear whether this activation was aresult of preventing denaturation or if the albumin affected theavailability of the substrate. A preferable formulation of human sHASEGPcould therefore include Albumin and a metal salt consisting of eitherCalcium or Magnesium.

Example-15 Spreading Activity of Purified sHASEGP In Vivo

a. Protocol

Purified sHASEGP in 10 mM Hepes PH 7.4, 150 mM NaCl 0.1% Pluronic wasdiluted to 0.5 U/ul in pyrogen free water with 0.15M NaCl. A series ofdilutions in 20 ul final of Saline were made to give a total of 0.01,0.05, 0.1 Units per injection. 20 ul of Trypan Blue solution was addedto a final volume of 40 ul and injected subcutaneously into the lateralskin on each side of balb ^(Nu/Nu) mice that had been previouslyanesthetized i.p. by ketamine/xylazine administration. Dye areas weremeasured in 2 dimensions with a microcaliper from t=0 to t=45 min. Areawas represented as mm². As a control recombinant Human HYAL1 that lacksneutral activity but is secreted was included.

b. Results

TEST ARTICLE DYE AREA @ 45 MIN A. Saline Control  51.5 mm² B. sHASEGP0.01 U  76.8 mm² C. sHASEGP 0.05 U 98.22 mm² D. sHASEGP 0.10 U 180.4 mm²E. HYAL1 100 U 67.48 mm²

Example-16 Kinetics of sHASEGP Diffusion Activity

a. Protocol

Recombinant purified sHASEGP_(HIS6) was separated into 2 aliquots. Onewas heated to 95 C for 15 minutes in a thermocycler with a heated lid.The other remained at room temperature. Thermal inactivation of enzymeactivity was verified in the microtiter based enzyme assay. For kineticanalysis heat inactivated verses native material was tested. 4 Units ofpurified sHASEGP or equivalent heat inactivated material was injectedsubcutaneously with trypan blue dye. Areas were tested at various timepoints up to 15 minutes.

b. Results

4 UNITS 4 UNITS HEAT INACTIVATED t_(minute post injection)t_(minute post injection) t₀ = 52.38 t₀ = 50.58 t₃ = 116.51 t₃ = 65.48t_(6.5) = 181.93 T_(6.5) = 63.87 t₁₀ = 216.96 T₁₀ = 65.80 t₁₆ = 279.99T₁₆ = 74.3

Example-17 Restoration of the Dermal Barrier Broken Down by sHASEGP

a. Protocol

To establish the regeneration time of the pores opened with sHASEGPfollowing subcutaneous administration, 2 Units of purified sHASEGP orsaline control was injected into two opposing lateral sitessubcutaneously in animals at t=0 followed by injection with trypan blueat the same site at 30 min 60 min and 24 hours. Area of the dyediffusion at t=15 minutes post injection was recorded for each timepoint compared to the control.

b. Results

2 UNITS SALINE CONTROL T_(hour post injection sHASEGP)t_(hour post injection sHASEGP) t_(0.5 h) = 183 t_(0.5 h) = 54 t_(1 hr)= 167 t_(1 hr) = 50 t_(22 hr) = 61 t_(22 hr) = 48

The results demonstrate that the dermal barrier reconstitutes within 24hours of administration of 2 Units of enzyme.

Example-18 Determination of the Size of Channels Opened by sHASEGP

It was shown that human sHASEGP opened channels in the interstitialspace sufficient to permit the diffusion of a small molecule, i.e.trypan blue dye. However, it was unknown what the upper limits were onthe size of particles that could diffuse in the presence of sHASEGP.

a. Protocol

Florescent molecules of varying sizes were used to determine the size ofthe channels opened by human sHASEGP, Flouresceinated Dextrans of 4,400and 2 million Da Average Molecular Weight (Sigma) as well as flouresceinlabeled beads of defined diameters from 20 nanometers to 500 nanometers(Molecular Probes), were administered subcutaneously in a volume of 40ul with following injection of sHASEGP or saline control in the samesites. Area of the dye front was then measured in two dimensions at 15minutes post injection.

b. Results

Diffusion Test Area at Diffusion Agent Particle Size 15 min Stand DevsHASEGP 4400 Da 84.2 25.7 Control 4400 Da 38.0 5.8 sHASEGP 2 × 10E6 Da141.2 4.5 Control 2 × 10E6 Da 51.7 8.1 sHASEGP  20 nm Diameter 92.3 20.6Control  20 nm Diameter 51.6 3.0 sHASEGP 100 nm Diameter 61.0 5.7Control 100 nm Diameter 40.0 7.0 sHASEGP 200 nm Diameter 35.5 1.6Control 200 nm Diameter 27.9 8.2 sHASEGP 500 nm Diameter 44.8 13.6Control 500 nm Diameter 41.2 9.8

The results demonstrated that molecules from approximately 1 kDa (TrypanBlue) to 50 nm in diameter (Latex Beads) showed enhanced diffusionfollowing administration of sHASEGP. While bovine serum albumin (66 kDA)showed similar kinetics of diffusion to trypan blue, the 50 nm latexbeads required significantly more time to diffuse. 500 nm beads showedno diffusion up to 480 minutes.

Example-19 Serum Pharmacokinetics Profiles of Biotinylated AntibodiesFollowing Subcutaneous Co-Injection of Human sHASEGP

a. Protocol

Female Balb/c mice were anesthetized with a mixture ofketamine/xylazine. The mice were then injected subcutaneously with 20 ulof 0.5 mg/ml solution of biotinylated mouse IgG mixed with 20 ul ofeither saline or 20 ul sHASEGP containing 4 Units of activity.

b. Results

TIME POST INJECTION CONTROL sHASEGP (4 U) Serum IgG t = 0 hrs 0 ng/ml  0 ng/ml Serum IgG t = 2 hrs 0 ng/ml  360 ng/ml Serum IgG t = 51 hrs4152 ng/ml   4176 ng/ml

The results demonstrate that sHASEGP increases the kinetics of serumdistribution of large molecules in circulation. Where no biotinylatedIgG could be detected in the control group at 2 hours, 360 ng/ml wasapparent by 2 hours in the sHASEGP group.

Example-20 Spreading Activity of Subcutaneously Injected MoleculesFollowing Intravenous Injection of Human sHASEGP

a. Protocol

Four sites for dye injection were utilized per dose of each Test Articleand carrier control. Dye injection was 45 minutes after i.v. injection.Each dose of test or control article was injected i.v. into 2 animals.Measurement of the dye front area post 45 minute enzyme administrationwas calculated at 2.5, 5, 10 and 15 minutes for each dose or carriercontrol.

b. Results

Results demonstrated that highly purified sHASEGP was systemicallyavailable to distal tissues upon intravenous administration. Thespreading activity of systemically administered sHASEGP was dosedependent, with a 10 unit injection being indistinguishable from carriercontrol.

Dose Time Mean Area Type IV Minutes (mm²) SD PH20 1000 2.5 86.4172.83419345 minute enzyme administration was calculated at 2.5, 5, 10 and 15minutes for each dose or carrier control.

b. Results

Results demonstrated that highly purified sHASEGP was systemicallyavailable to distal tissues upon intravenous administration. Thespreading activity of systemically administered sHASEGP was dosedependent, with a 10 unit injection being indistinguishable from carriercontrol.

Dose Time Mean Area Type IV Minutes (mm²) SD PH20 1000 2.5 86.4172.834193 PH20 1000 5 102.17 2.221146 PH20 1000 10 124.53 6.304944 PH201000 15 129.81 1.434319 PH20 300 2.5 59.137 7.218615 PH20 300 5 73.6387.51197 PH20 300 10 87.092 8.686008 PH20 300 15 92.337 10.66466 PH20 1002.5 56.308 7.741934 PH20 100 5 63.156 11.42052 PH20 100 10 76.51916.18449 PH20 100 15 77.432 17.32264 PH20 30 2.5 50.534 10.64287 PH20 305 59.493 5.163971 PH20 30 10 68.102 11.00071 PH20 30 15 71.118 9.934212PH20 10 2.5 36.4 3.807072 PH20 10 5 39.859 6.680932 PH20 10 10 45.6494.44936 PH20 10 15 48.41 6.546835 Control 0 2.5 34.652 5.935037 Control0 5 36.279 3.614544 Control 0 10 44.687 5.821216 Control 0 15 53.0022.812439

Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.

What is claimed:
 1. A method for reducing intraocular pressure in theeye of a subject, comprising administering a composition comprising ahyaluronidase polypeptide to the eye, wherein: the hyaluronidasepolypeptide contains at least one sugar moiety that is covalentlyattached to an asparagine residue of the hyaluronidase polypeptide; thehyaluronidase polypeptide is catalytically active; the hyaluronidasepolypeptide is secreted; and the hyaluronidase polypeptide consists of asequence of amino acids that has at least 98% amino acid sequenceidentity with the sequence of amino acids set forth as amino acidresidues 36-483 of SEQ ID NO:1.
 2. The method of claim 1, wherein thehyaluronidase polypeptide has at least 99% amino acid sequence identitywith the sequence of amino acids set forth as amino acid residues 36-483of SEQ ID NO:1.
 3. A method for reducing intraocular pressure in the eyeof a subject, comprising administering a composition comprising ahyaluronidase polypeptide to the eye, wherein: the hyaluronidasepolypeptide contains at least one sugar moiety that is covalentlyattached to an asparagine residue of the hyaluronidase polypeptide; thehyaluronidase polypeptide is catalytically active; the hyaluronidasepolypeptide is secreted; and the hyaluronidase polypeptide either: (i)consists of the sequence of amino acids set forth as amino acids 36-477,36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1; or (ii)has at least 95% sequence identity with the sequence of amino acids setforth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or36-483 of SEQ ID NO:1, wherein the amino acid sequence of thehyaluronidase polypeptide differs from the sequence of amino acids setforth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or36-483 of SEQ ID NO: 1 only by amino acid substitutions.
 4. The methodof claim 1, wherein the hyaluronidase polypeptide consists of residues36-485 of SEQ ID NO:1.
 5. The method of claim 1, wherein thehyaluronidase polypeptide is secreted when produced in CHO cells.
 6. Themethod of claim 1, wherein the C-terminal residue of the hyaluronidasepolypeptide in the composition is at a position corresponding toposition 477, 478, 479, 480, 481, 482 or 483 of SEQ ID NO:1.
 7. Themethod of claim 1, wherein the sequence of amino acid residues of thehyaluronidase polypeptide in the composition consists of residues36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1.8. The method of claim 1, wherein the hyaluronidase polypeptide ischemically linked to a polymer.
 9. The method of claim 3, wherein thehyaluronidase polypeptide is chemically linked to a polymer.
 10. Themethod of claim 8, wherein the polymer is a polyethylene glycol (PEG) ora dextran.
 11. The method of claim 10, wherein the polymer is a PEG. 12.The method of claim 9, wherein the polymer is a polyethylene glycol(PEG) or a dextran.
 13. The method of claim 1, wherein the subject hasglaucoma or has elevated intraocular pressure associated with cataractsor treatment of cataracts.
 14. The method of claim 3, wherein thesubject has glaucoma or has elevated intraocular pressure associatedwith cataracts or treatment of cataracts.
 15. The method of claim 13,wherein the subject has been treated with viscoelastic substances. 16.The method of claim 13, wherein the subject has been treated withhyaluronic acid.
 17. The method of claim 13, wherein the subject hasopen angle glaucoma.
 18. The method of claim 1, wherein the subject hasvitreous aggregates, vitreous hemorrhage, macular degeneration ordiabetic retinopathy.
 19. The method of claim 1, wherein the compositioncomprising the hyaluronidase polypeptide is co-administered with otherenzymes that promote reshaping of the cornea by corrective contactlenses.
 20. The method of claim 1, wherein the composition comprisingthe hyaluronidase polypeptide is administered intraocularly.
 21. Themethod of claim 3, wherein the subject has vitreous aggregates, vitreoushemorrhage, macular degeneration or diabetic retinopathy.
 22. The methodof claim 3, wherein the composition comprising the hyaluronidasepolypeptide is co-administered with other enzymes that promote reshapingof the cornea by corrective contact lenses.
 23. The method of claim 3,wherein the composition comprising the hyaluronidase polypeptide isadministered intraocularly.
 24. The method of claim 3, wherein thehyaluronidase polypeptide consists of the sequence of amino acids setforth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or36-483 of SEQ ID NO:1.